register interest

Professor E. Yvonne Jones FRS FMedSci

Research Area: Protein Science and Structural Biology
Technology Exchange: Bioinformatics, Crystallography, Drug discovery, Microscopy (Confocal), Microscopy (EM) and Protein interaction
Scientific Themes: Protein Science & Structural Biology and Cancer Biology
Keywords: structural biology, tumour immunology, developmental biology, protein-protein interactions, signalling complexes and x-ray crystallography
Web Links:
The structure of the RPTPmu trans dimer (Aricescu et al Science 2007)

The structure of the RPTPmu trans dimer (Aricescu et al Science 2007)

Semaphorin-plexin recognition complex (Janssen et al Nature 2010)

Semaphorin-plexin recognition complex (Janssen et al Nature 2010)

Model for opposing RPTPsigma-proteoglycan functions (Coles et al Science 2011)

Model for opposing RPTPsigma-proteoglycan functions (Coles et al Science 2011)

Yvonne Jones is Director of the Cancer Research UK Receptor Structure Research Group which is focused on the structural biology of extracellular recognition and signalling complexes. The group's core techniques include protein crystallography and, increasingly, cryo electron microscopy, which are used to generate high resolution structural information. Importantly, studies using these techniques are integrated with advanced light microscopy and cryo electron tomography, as well as cell-based functional studies, to probe molecular mechanisms at the cell surface.

The group's research addresses fundamental questions about cell-cell signalling systems of importance to human health. How are signalling assemblies arranged? Which features are necessary for normal signal transduction into the cell? What mechanisms trigger dysfunctional signalling? The work ties into an extensive network of interdisciplinary local and international collaborations with the ultimate aim of learning how to manipulate these signalling systems for the design of new clinical therapies. Current projects within the group focus on signalling systems of importance in developmental biology. These include systems involved in cell guidance (e.g. semaphorin/plexin and ephrin/Eph signalling), as well as mechanisms controlling signalling by the Wnt family of morphogens.

Name Department Institution Country
Professor Sir Andrew J McMichael NDM Research Building Oxford University, NDM Research Building United Kingdom
Prof Vincenzo Cerundolo (RDM) Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Prof Lars Fugger (RDM) Weatherall Institute of Molecular Medicine Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Schwerd T, Twigg SRF, Aschenbrenner D, Manrique S, Miller KA, Taylor IB, Capitani M, McGowan SJ, Sweeney E, Weber A et al. 2017. A biallelic mutation in IL6ST encoding the GP130 co-receptor causes immunodeficiency and craniosynostosis. J Exp Med, 214 (9), pp. 2547-2562. | Show Abstract | Read more

Multiple cytokines, including interleukin 6 (IL-6), IL-11, IL-27, oncostatin M (OSM), and leukemia inhibitory factor (LIF), signal via the common GP130 cytokine receptor subunit. In this study, we describe a patient with a homozygous mutation of IL6ST (encoding GP130 p.N404Y) who presented with recurrent infections, eczema, bronchiectasis, high IgE, eosinophilia, defective B cell memory, and an impaired acute-phase response, as well as skeletal abnormalities including craniosynostosis. The p.N404Y missense substitution is associated with loss of IL-6, IL-11, IL-27, and OSM signaling but a largely intact LIF response. This study identifies a novel immunodeficiency with phenotypic similarities to STAT3 hyper-IgE syndrome caused by loss of function of GP130.

Zebisch M, Jackson VA, Zhao Y, Jones EY. 2016. Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf. Structure, 24 (9), pp. 1599-1605. | Show Abstract | Read more

Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling. We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains. The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor. We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD. DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC. Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.

Seiradake E, Jones EY, Klein R. 2016. Structural Perspectives on Axon Guidance. Annu Rev Cell Dev Biol, 32 (1), pp. 577-608. | Show Abstract | Read more

Axon guidance relies on a combinatorial code of receptor and ligand interactions that direct adhesive/attractive and repulsive cellular responses. Recent structural data have revealed many of the molecular mechanisms that govern these interactions and enabled the design of sophisticated mutant tools to dissect their biological functions. Here, we discuss the structure/function relationships of four major classes of guidance cues (ephrins, semaphorins, slits, netrins) and examples of morphogens (Wnt, Shh) and of cell adhesion molecules (FLRT). These cell signaling systems rely on specific modes of receptor-ligand binding that are determined by selective binding sites; however, defined structure-encoded receptor promiscuity also enables cross talk between different receptor/ligand families and can also involve extracellular matrix components. A picture emerges in which a multitude of highly context-dependent structural assemblies determines the finely tuned cellular behavior required for nervous system development.

Kong Y, Janssen BJ, Malinauskas T, Vangoor VR, Coles CH, Kaufmann R, Ni T, Gilbert RJ, Padilla-Parra S, Pasterkamp RJ, Jones EY. 2016. Structural Basis for Plexin Activation and Regulation. Neuron, 91 (3), pp. 548-560. | Show Abstract | Read more

Class A plexins (PlxnAs) act as semaphorin receptors and control diverse aspects of nervous system development and plasticity, ranging from axon guidance and neuron migration to synaptic organization. PlxnA signaling requires cytoplasmic domain dimerization, but extracellular regulation and activation mechanisms remain unclear. Here we present crystal structures of PlxnA (PlxnA1, PlxnA2, and PlxnA4) full ectodomains. Domains 1-9 form a ring-like conformation from which the C-terminal domain 10 points away. All our PlxnA ectodomain structures show autoinhibitory, intermolecular "head-to-stalk" (domain 1 to domain 4-5) interactions, which are confirmed by biophysical assays, live cell fluorescence microscopy, and cell-based and neuronal growth cone collapse assays. This work reveals a 2-fold role of the PlxnA ectodomains: imposing a pre-signaling autoinhibitory separation for the cytoplasmic domains via intermolecular head-to-stalk interactions and supporting dimerization-based PlxnA activation upon ligand binding. More generally, our data identify a novel molecular mechanism for preventing premature activation of axon guidance receptors.

Chang VT, Fernandes RA, Ganzinger KA, Lee SF, Siebold C, McColl J, Jönsson P, Palayret M, Harlos K, Coles CH et al. 2016. Initiation of T cell signaling by CD45 segregation at 'close contacts'. Nat Immunol, 17 (5), pp. 574-582. | Show Abstract | Read more

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.

Chavent M, Seiradake E, Jones EY, Sansom MS. 2016. Structures of the EphA2 Receptor at the Membrane: Role of Lipid Interactions. Structure, 24 (2), pp. 337-347. | Show Abstract | Read more

Ephs are transmembrane receptors that mediate cell-cell signaling. The N-terminal ectodomain binds ligands and enables receptor clustering, which activates the intracellular kinase. Relatively little is known about the function of the membrane-proximal fibronectin domain 2 (FN2) of the ectodomain. Multiscale molecular dynamics simulations reveal that FN2 interacts with lipid bilayers via a site comprising K441, R443, R465, Q462, S464, S491, W467, F490, and P459-461. FN2 preferentially binds anionic lipids, a preference that is reduced in the mutant K441E + R443E. We confirm these results by measuring the binding of wild-type and mutant FN2 domains to lipid vesicles. In simulations of the complete EphA2 ectodomain plus the transmembrane region, we show that FN2 anchors the otherwise flexible ectodomain at the surface of the bilayer. Altogether, our data suggest that FN2 serves a dual function of interacting with anionic lipids and constraining the structure of the EphA2 ectodomain to adopt membrane-proximal configurations.

Zebisch M, Jones EY. 2015. Crystal structure of R-spondin 2 in complex with the ectodomains of its receptors LGR5 and ZNRF3. J Struct Biol, 191 (2), pp. 149-155. | Show Abstract | Read more

The four secreted R-spondin (Rspo1-4) proteins of vertebrates function as stem cell growth factors and potentiate canonical Wnt signalling. Rspo proteins act by cross-linking members of two cell surface receptor families, complexing the stem cell markers LGR4-6 with the Frizzled-specific E3 ubiquitin ligases ZNRF3/RNF43. The consequent internalisation of the ternary LGR-Rspo-E3 complex removes the E3 ligase activity, which otherwise targets the Wnt receptor Frizzled for degradation, and thus enhances Wnt signalling. Multiple combinations of LGR4-6, Rspo1-4 and ZNRF3/RNF43 are possible, implying the existence of generic interaction determinants, but also of specific differences in complex architecture and activity. We present here a high resolution crystal structure of an ectodomain variant of human LGR5 (hLGR5ecto) complexed with a signalling competent fragment of mouse Rspo2 (mRspo2Fu1-Fu2). The structure shows that the particularly potent Rspo2 ligand engages LGR5 in a fashion almost identical to that reported for hRSPO1. Comparison of our hLGR5ecto structure with previously published structures highlights a surprising plasticity of the LGR ectodomains, characterised by a nearly 9° or larger rotation of the N-terminal half of the horseshoe-like fold relative to the C-terminal half. We also report a low resolution hLGR5-mRspo2Fu1-Fu2-mZNRF3ecto ternary complex structure. This crystal structure confirms our previously suggested hypothesis, showing that Rspo proteins cross-link LGRs and ZNRF3 into a 2:2:2 complex, whereas a 1:1:1 complex is formed with RNF43.

Zebisch M, Jones EY. 2015. ZNRF3/RNF43--A direct linkage of extracellular recognition and E3 ligase activity to modulate cell surface signalling. Prog Biophys Mol Biol, 118 (3), pp. 112-118. | Show Abstract | Read more

The interactions of extracellular ligands with single membrane spanning receptors, such as kinases, typically serve to agonise or antagonise the intracellular activation of signalling pathways. Within the cell, E3 ligases can act to alter the localisation and activity of proteins involved in signalling systems. Structural and functional characterisation of two closely related single membrane spanning molecules, RNF43 and ZNRF3, has recently revealed the receptor-like functionalities of a ligand-binding ectodomain combined with the intracellular architecture and activity of an E3 ligase. This direct link provides a hereto novel mechanism for extracellular control of ubiquitin ligase activity that is used for the modulation of Wnt signalling, a pathway of major importance in embryogenesis, stem cell biology and cancer. In this review we discuss recent findings for the structure and interactions of the extracellular region of RNF43/ZNRF3 and draw parallels with the properties and function of signalling receptor ectodomains.

Jones EY. 2015. Understanding cell signalling systems: paving the way for new therapies. Philos Trans A Math Phys Eng Sci, 373 (2036), pp. 20130155-20130155. | Show Abstract | Read more

The cell-to-cell signalling mechanisms of multi-cellular organisms orchestrate human development during embryogenesis and control homeostasis in adult tissues. These are mechanisms vital to human health and perturbation of cell-to-cell signalling is a contributing factor in many pathologies including cancer. The semaphorin cell guidance cues and their cognate plexin receptors exemplify a cell-to-cell signalling system for which insights into mechanistic principles are emerging. X-ray crystallographic data from Diamond beam lines have enabled us to probe the inner workings of semaphorin-plexin signalling to atomic-level resolutions. Importantly, we can complement protein crystallographic results with biophysical and cellular studies to dovetail structural information with functional impact. The signature seven-bladed β propeller 'sema' domain of the semaphorins forms a dimer; in contrast the equivalent domain in the plexins is monomeric. The generic architecture of a semaphorin-plexin complex is characterized by the dimeric semaphorin cross-linking two copies of the plexin receptor. For specific family members, the co-receptor neuropilin serves to bolster this architecture, but in all cases, the dimeric interaction lies at the core of the ligand receptor complex, providing the essential trigger for signalling.

Kakugawa S, Langton PF, Zebisch M, Howell S, Chang TH, Liu Y, Feizi T, Bineva G, O'Reilly N, Snijders AP et al. 2015. Notum deacylates Wnt proteins to suppress signalling activity. Nature, 519 (7542), pp. 187-192. | Show Abstract | Read more

Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase.

Zhang X, Cheong SM, Amado NG, Reis AH, MacDonald BT, Zebisch M, Jones EY, Abreu JG, He X. 2015. Notum is required for neural and head induction via Wnt deacylation, oxidation, and inactivation. Dev Cell, 32 (6), pp. 719-730. | Show Abstract | Read more

Secreted Wnt morphogens are essential for embryogenesis and homeostasis and require a lipid/palmitoleoylate modification for receptor binding and activity. Notum is a secreted Wnt antagonist that belongs to the α/β hydrolase superfamily, but its mechanism of action and roles in vertebrate embryogenesis are not fully understood. Here, we report that Notum hydrolyzes the Wnt palmitoleoylate adduct extracellularly, resulting in inactivated Wnt proteins that form oxidized oligomers incapable of receptor binding. Thus, Notum is a Wnt deacylase, and palmitoleoylation is obligatory for the Wnt structure that maintains its active monomeric conformation. Notum is expressed in naive ectoderm and neural plate in Xenopus and is required for neural and head induction. These findings suggest that Notum is a prerequisite for the "default" neural fate and that distinct mechanisms of Wnt inactivation by the Tiki protease in the Organizer and the Notum deacylase in presumptive neuroectoderm orchestrate vertebrate brain development.

Chang TH, Hsieh FL, Zebisch M, Harlos K, Elegheert J, Jones EY. 2015. Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan. Elife, 4 (JULY 2015), pp. 1-27. | Show Abstract | Read more

Wnt signalling regulates multiple processes including angiogenesis, inflammation, and tumorigenesis. Norrin (Norrie Disease Protein) is a cystine-knot like growth factor. Although unrelated to Wnt, Norrin activates the Wnt/β-catenin pathway. Signal complex formation involves Frizzled4 (Fz4), low-density lipoprotein receptor related protein 5/6 (Lrp5/6), Tetraspanin-12 and glycosaminoglycans (GAGs). Here, we report crystallographic and small-angle X-ray scattering analyses of Norrin in complex with Fz4 cysteine-rich domain (Fz4CRD), of this complex bound with GAG analogues, and of unliganded Norrin and Fz4CRD. Our structural, biophysical and cellular data, map Fz4 and putative Lrp5/6 binding sites to distinct patches on Norrin, and reveal a GAG binding site spanning Norrin and Fz4CRD. These results explain numerous disease-associated mutations. Comparison with the Xenopus Wnt8-mouse Fz8CRD complex reveals Norrin mimics Wnt for Frizzled recognition. The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling.

Johnson E, Seiradake E, Jones EY, Davis I, Grünewald K, Kaufmann R. 2015. Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins. Sci Rep, 5 (1), pp. 9583. | Show Abstract | Read more

We introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge. We identified key aspects of the sample preparation procedure of high pressure freezing, freeze substitution and resin embedding that are critical for preserving fluorescence and photo-switching of standard fluorescent proteins, such as mGFP, mVenus and mRuby2. This enabled us to combine single molecule localization microscopy with transmission electron microscopy imaging of standard fluorescent proteins in cryo-fixed resin embedded cells. We achieved a structural resolution of 40-50 nm (~17 nm average single molecule localization accuracy) in the fluorescence images without the use of chemical fixation or special fluorophores. Using this approach enabled the correlation of fluorescently labeled structures to the ultrastructure in the same cell at the nanometer level and superior structural preservation.

Srivastava M, Duan G, Kershaw NJ, Athanasopoulos V, Yeo JH, Ose T, Hu D, Brown SH, Jergic S, Patel HR et al. 2015. Roquin binds microRNA-146a and Argonaute2 to regulate microRNA homeostasis. Nat Commun, 6 pp. 6253. | Show Abstract | Read more

Roquin is an RNA-binding protein that prevents autoimmunity and inflammation via repression of bound target mRNAs such as inducible costimulator (Icos). When Roquin is absent or mutated (Roquin(san)), Icos is overexpressed in T cells. Here we show that Roquin enhances Dicer-mediated processing of pre-miR-146a. Roquin also directly binds Argonaute2, a central component of the RNA-induced silencing complex, and miR-146a, a microRNA that targets Icos mRNA. In the absence of functional Roquin, miR-146a accumulates in T cells. Its accumulation is not due to increased transcription or processing, rather due to enhanced stability of mature miR-146a. This is associated with decreased 3' end uridylation of the miRNA. Crystallographic studies reveal that Roquin contains a unique HEPN domain and identify the structural basis of the 'san' mutation and Roquin's ability to bind multiple RNAs. Roquin emerges as a protein that can bind Ago2, miRNAs and target mRNAs, to control homeostasis of both RNA species.

Seiradake E, Zhao Y, Lu W, Aricescu AR, Jones EY. 2015. Production of cell surface and secreted glycoproteins in mammalian cells. Methods Mol Biol, 1261 pp. 115-127. | Show Abstract | Read more

Mammalian protein expression systems are becoming increasingly popular for the production of eukaryotic secreted and cell surface proteins. Here we describe methods to produce recombinant proteins in adherent or suspension human embryonic kidney cell cultures, using transient transfection or stable cell lines. The protocols are easy to scale up and cost-efficient, making them suitable for protein crystallization projects and other applications that require high protein yields.

Delloye-Bourgeois C, Jacquier A, Charoy C, Reynaud F, Nawabi H, Thoinet K, Kindbeiter K, Yoshida Y, Zagar Y, Kong Y et al. 2015. PlexinA1 is a new Slit receptor and mediates axon guidance function of Slit C-terminal fragments. Nat Neurosci, 18 (1), pp. 36-45. | Show Abstract | Read more

Robo-Slit and Plexin-Semaphorin signaling participate in various developmental and pathogenic processes. During commissural axon guidance in the spinal cord, chemorepulsion by Semaphorin3B and Slits controls midline crossing. Slit processing generates an N-terminal fragment (SlitN) that binds to Robo1 and Robo2 receptors and mediates Slit repulsive activity, as well as a C-terminal fragment (SlitC) with an unknown receptor and bioactivity. We identified PlexinA1 as a Slit receptor and found that it binds the C-terminal Slit fragment specifically and transduces a SlitC signal independently of the Robos and the Neuropilins. PlexinA1-SlitC complexes are detected in spinal cord extracts, and ex vivo, SlitC binding to PlexinA1 elicits a repulsive commissural response. Analysis of various ligand and receptor knockout mice shows that PlexinA1-Slit and Robo-Slit signaling have complementary roles during commissural axon guidance. Thus, PlexinA1 mediates both Semaphorin and Slit signaling, and Slit processing generates two active fragments, each exerting distinct effects through specific receptors.

Malinauskas T, Jones EY. 2014. Extracellular modulators of Wnt signalling. Curr Opin Struct Biol, 29 pp. 77-84. | Show Abstract | Read more

Wnt morphogens are secreted signalling proteins that play leading roles in embryogenesis and tissue homeostasis throughout life. Wnt signalling is controlled by multiple mechanisms, including posttranslational modification of Wnts, antagonist binding (to Wnts or their receptors), and regulation of the availability of Wnt receptors. Recent crystallographic, structure-guided biophysical and cell-based studies have advanced our understanding of how Wnt signalling is regulated at the cell surface. Structures include Wnt in complex with the cysteine-rich domain (CRD) of Frizzled, extracellular fragments of Wnt co-receptor LRP6, LRP6-binding antagonists Dickkopf and Sclerostin, antagonists 5T4/WAIF1 and Wnt inhibitory factor 1 (WIF-1), as well as Frizzled-ubiquitin ligases ZNRF3/RNF43 (in isolation and in complexes with Wnt signalling promoters R-spondins and LGR5). We review recent discoveries and remaining questions.

Seiradake E, del Toro D, Nagel D, Cop F, Härtl R, Ruff T, Seyit-Bremer G, Harlos K, Border EC, Acker-Palmer A et al. 2014. FLRT structure: balancing repulsion and cell adhesion in cortical and vascular development. Neuron, 84 (2), pp. 370-385. | Show Abstract | Read more

FLRTs are broadly expressed proteins with the unique property of acting as homophilic cell adhesion molecules and as heterophilic repulsive ligands of Unc5/Netrin receptors. How these functions direct cell behavior and the molecular mechanisms involved remain largely unclear. Here we use X-ray crystallography to reveal the distinct structural bases for FLRT-mediated cell adhesion and repulsion in neurons. We apply this knowledge to elucidate FLRT functions during cortical development. We show that FLRTs regulate both the radial migration of pyramidal neurons, as well as their tangential spread. Mechanistically, radial migration is controlled by repulsive FLRT2-Unc5D interactions, while spatial organization in the tangential axis involves adhesive FLRT-FLRT interactions. Further, we show that the fundamental mechanisms of FLRT adhesion and repulsion are conserved between neurons and vascular endothelial cells. Our results reveal FLRTs as powerful guidance factors with structurally encoded repulsive and adhesive surfaces.

Coles CH, Jones EY, Aricescu AR. 2015. Extracellular regulation of type IIa receptor protein tyrosine phosphatases: mechanistic insights from structural analyses. Semin Cell Dev Biol, 37 pp. 98-107. | Show Abstract | Read more

The receptor protein tyrosine phosphatases (RPTPs) exhibit a wide repertoire of cellular signalling functions. In particular, type IIa RPTP family members have recently been highlighted as hubs for extracellular interactions in neurons, regulating neuronal extension and guidance, as well as synaptic organisation. In this review, we will discuss the recent progress of structural biology investigations into the architecture of type IIa RPTP ectodomains and their interactions with extracellular ligands. Structural insights, in combination with biophysical and cellular studies, allow us to begin to piece together molecular mechanisms for the transduction and integration of type IIa RPTP signals and to propose hypotheses for future experimental validation.

Kaufmann R, Schellenberger P, Seiradake E, Dobbie IM, Jones EY, Davis I, Hagen C, Grünewald K. 2014. Super-resolution microscopy using standard fluorescent proteins in intact cells under cryo-conditions. Nano Lett, 14 (7), pp. 4171-4175. | Show Abstract | Read more

We introduce a super-resolution technique for fluorescence cryo-microscopy based on photoswitching of standard genetically encoded fluorescent marker proteins in intact mammalian cells at low temperature (81 K). Given the limit imposed by the lack of cryo-immersion objectives, current applications of fluorescence cryo-microscopy to biological specimens achieve resolutions between 400-500 nm only. We demonstrate that the single molecule characteristics of reversible photobleaching of mEGFP and mVenus at liquid nitrogen temperature are suitable for the basic concept of single molecule localization microscopy. This enabled us to perform super-resolution imaging of vitrified biological samples and to visualize structures in unperturbed fast frozen cells for the first time with a structural resolution of ∼125 nm (average single molecule localization accuracy ∼40 nm), corresponding to a 3-5 fold resolution improvement.

Zhao Y, Malinauskas T, Harlos K, Jones EY. 2014. Structural insights into the inhibition of Wnt signaling by cancer antigen 5T4/Wnt-activated inhibitory factor 1 Structure, 22 (4), pp. 612-620. | Show Abstract | Read more

The tumor antigen 5T4/WAIF1 (Wnt-activated inhibitory factor 1; also known as Trophoblast glycoprotein TPBG) is a cell surface protein targeted in multiple cancer immunotherapy clinical trials. Recently, it has been shown that 5T4/WAIF1 inhibits Wnt/β-catenin signaling, a signaling system central to many developmental and pathological processes. Wnt/β-catenin signaling is controlled by multiple inhibitors and activators. Here, we report crystal structures for the extracellular domain of 5T4/WAIF1 at 1.8 Å resolution. They reveal a highly glycosylated, rigid core, comprising eight leucine-rich repeats (LRRs), which serves as a platform to present evolutionarily conserved surface residues in the N-terminal LRR1. Structural and cell-based analyses, coupled with previously reported in vivo data, suggest that Tyr325 plus the LRR1 surface centered on a second exposed aromatic residue, Phe97, are essential for inhibition of Wnt/β-catenin signaling. These results provide a structural basis for the development of 5T4/WAIF1-targeted therapies that preserve or block 5T4/WAIF1-mediated inhibition of Wnt/β-catenin signaling. © 2014 The Authors.

Zhao Y, Malinauskas T, Harlos K, Jones EY. 2014. Structural insights into the inhibition of Wnt signaling by cancer antigen 5T4/Wnt-activated inhibitory factor 1. Structure, 22 (4), pp. 612-620. | Show Abstract | Read more

The tumor antigen 5T4/WAIF1 (Wnt-activated inhibitory factor 1; also known as Trophoblast glycoprotein TPBG) is a cell surface protein targeted in multiple cancer immunotherapy clinical trials. Recently, it has been shown that 5T4/WAIF1 inhibits Wnt/β-catenin signaling, a signaling system central to many developmental and pathological processes. Wnt/β-catenin signaling is controlled by multiple inhibitors and activators. Here, we report crystal structures for the extracellular domain of 5T4/WAIF1 at 1.8 Å resolution. They reveal a highly glycosylated, rigid core, comprising eight leucine-rich repeats (LRRs), which serves as a platform to present evolutionarily conserved surface residues in the N-terminal LRR1. Structural and cell-based analyses, coupled with previously reported in vivo data, suggest that Tyr325 plus the LRR1 surface centered on a second exposed aromatic residue, Phe97, are essential for inhibition of Wnt/β-catenin signaling. These results provide a structural basis for the development of 5T4/WAIF1-targeted therapies that preserve or block 5T4/WAIF1-mediated inhibition of Wnt/β-catenin signaling.

Salio M, Silk JD, Jones EY, Cerundolo V. 2014. Biology of CD1- and MR1-restricted T cells. Annu Rev Immunol, 32 (1), pp. 323-366. | Show Abstract | Read more

Over the past 15 years, investigators have shown that T lymphocytes can recognize not only peptides in the context of MHC class I and class II molecules but also foreign and self-lipids in association with the nonclassical MHC class I-like molecules, CD1 proteins. In this review, we describe the most recent events in the field, with particular emphasis on (a) structural and functional aspects of lipid presentation by CD1 molecules, (b) the development of CD1d-restricted invariant natural killer T (iNKT) cells and transcription factors required for their differentiation, (c) the ability of iNKT cells to modulate innate and adaptive immune responses through their cross talk with lymphoid and myeloid cells, and (d) MR1-restricted and group I (CD1a, CD1b, and CD1c)-restricted T cells.

Baud A, Guryev V, Hummel O, Johannesson M, Rat Genome Sequencing and Mapping Consortium, Flint J. 2014. Genomes and phenomes of a population of outbred rats and its progenitors. Sci Data, 1 pp. 140011. | Show Abstract | Read more

Finding genetic variants that contribute to phenotypic variation is one of the main challenges of modern genetics. We used an outbred population of rats (Heterogeneous Stock, HS) in a combined sequence-based and genetic mapping analysis to identify sequence variants and genes contributing to complex traits of biomedical relevance. Here we describe the sequences of the eight inbred progenitors of the HS and the variants that segregate between them. We report the genotyping of 1,407 HS rats, and the collection from 2,006 rats of 195 phenotypic measures that are relevant to models of anxiety, type 2 diabetes, hypertension and osteoporosis. We make available haplotype dosages for the 1,407 genotyped rats, since genetic mapping in the HS is best carried out by reconstructing each HS chromosome as a mosaic of the progenitor genomes. Finally, we have deposited an R object that makes it easy to incorporate our sequence data into any genetic study of HS rats. Our genetic data are available for both Rnor3.4 and Rnor5.0 rat assemblies.

Coles CH, Mitakidis N, Zhang P, Elegheert J, Lu W, Stoker AW, Nakagawa T, Craig AM, Jones EY, Aricescu AR. 2014. Structural basis for extracellular cis and trans RPTPσ signal competition in synaptogenesis. Nat Commun, 5 pp. 5209. | Show Abstract | Read more

Receptor protein tyrosine phosphatase sigma (RPTPσ) regulates neuronal extension and acts as a presynaptic nexus for multiple protein and proteoglycan interactions during synaptogenesis. Unknown mechanisms govern the shift in RPTPσ function, from outgrowth promotion to synaptic organization. Here, we report crystallographic, electron microscopic and small-angle X-ray scattering analyses, which reveal sufficient inter-domain flexibility in the RPTPσ extracellular region for interaction with both cis (same cell) and trans (opposite cell) ligands. Crystal structures of RPTPσ bound to its postsynaptic ligand TrkC detail an interaction surface partially overlapping the glycosaminoglycan-binding site. Accordingly, heparan sulphate and heparin oligomers compete with TrkC for RPTPσ binding in vitro and disrupt TrkC-dependent synaptic differentiation in neuronal co-culture assays. We propose that transient RPTPσ ectodomain emergence from the presynaptic proteoglycan layer allows capture by TrkC to form a trans-synaptic complex, the consequent reduction in RPTPσ flexibility potentiating interactions with additional ligands to orchestrate excitatory synapse formation.

Bauza K, Malinauskas T, Pfander C, Anar B, Jones EY, Billker O, Hill AV, Reyes-Sandoval A. 2014. Efficacy of a Plasmodium vivax malaria vaccine using ChAd63 and modified vaccinia Ankara expressing thrombospondin-related anonymous protein as assessed with transgenic Plasmodium berghei parasites. Infect Immun, 82 (3), pp. 1277-1286. | Show Abstract | Read more

Plasmodium vivax is the world's most widely distributed malaria parasite and a potential cause of morbidity and mortality for approximately 2.85 billion people living mainly in Southeast Asia and Latin America. Despite this dramatic burden, very few vaccines have been assessed in humans. The clinically relevant vectors modified vaccinia virus Ankara (MVA) and the chimpanzee adenovirus ChAd63 are promising delivery systems for malaria vaccines due to their safety profiles and proven ability to induce protective immune responses against Plasmodium falciparum thrombospondin-related anonymous protein (TRAP) in clinical trials. Here, we describe the development of new recombinant ChAd63 and MVA vectors expressing P. vivax TRAP (PvTRAP) and show their ability to induce high antibody titers and T cell responses in mice. In addition, we report a novel way of assessing the efficacy of new candidate vaccines against P. vivax using a fully infectious transgenic Plasmodium berghei parasite expressing P. vivax TRAP to allow studies of vaccine efficacy and protective mechanisms in rodents. Using this model, we found that both CD8+ T cells and antibodies mediated protection against malaria using virus-vectored vaccines. Our data indicate that ChAd63 and MVA expressing PvTRAP are good preerythrocytic-stage vaccine candidates with potential for future clinical application.

Zebisch M, Xu Y, Krastev C, MacDonald BT, Chen M, Gilbert RJ, He X, Jones EY. 2013. Structural and molecular basis of ZNRF3/RNF43 transmembrane ubiquitin ligase inhibition by the Wnt agonist R-spondin. Nat Commun, 4 pp. 2787. | Show Abstract | Read more

The four R-spondin (Rspo) proteins are secreted agonists of Wnt signalling in vertebrates, functioning in embryogenesis and adult stem cell biology. Through ubiquitination and degradation of Wnt receptors, the transmembrane E3 ubiquitin ligase ZNRF3 and related RNF43 antagonize Wnt signalling. Rspo ligands have been reported to inhibit the ligase activity through direct interaction with ZNRF3 and RNF43. Here we report multiple crystal structures of the ZNRF3 ectodomain (ZNRF3(ecto)), a signalling-competent Furin1-Furin2 (Fu1-Fu2) fragment of Rspo2 (Rspo2(Fu1-Fu2)), and Rspo2(Fu1-Fu2) in complex with ZNRF3(ecto), or RNF43(ecto). A prominent loop in Fu1 clamps into equivalent grooves in the ZNRF3(ecto) and RNF43(ecto) surface. Rspo binding enhances dimerization of ZNRF3(ecto) but not of RNF43(ecto). Comparison of the four Rspo proteins, mutants and chimeras in biophysical and cellular assays shows that their signalling potency depends on their ability to recruit ZNRF3 or RNF43 via Fu1 into a complex with LGR receptors, which interact with Rspo via Fu2.

Cited:

36

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Seiradake E, Schaupp A, Del Toro Ruiz D, Kaufmann R, Mitakidis N, Harlos K, Aricescu AR, Klein R, Jones EY. 2013. Structurally encoded intraclass differences in EphA clusters drive distinct cell responses Nature Structural and Molecular Biology, 20 (8), pp. 958-964. | Show Abstract | Read more

Functional outcomes of ephrin binding to Eph receptors (Ephs) range from cell repulsion to adhesion. Here we used cell collapse and stripe assays, showing contrasting effects of human ephrinA5 binding to EphA2 and EphA4. Despite equivalent ligand binding affinities, EphA4 triggered greater cell collapse, whereas EphA2-expressing cells adhered better to ephrinA5-coated surfaces. Chimeric receptors showed that the ectodomain is a major determinant of cell response. We report crystal structures of EphA4 ectodomain alone and in complexes with ephrinB3 and ephrinA5. These revealed closed clusters with a dimeric or circular arrangement in the crystal lattice, contrasting with extended arrays previously observed for EphA2 ectodomain. Localization microscopy showed that ligand-stimulated EphA4 induces smaller clusters than does EphA2. Mutant Ephs link these characteristics to interactions observed in the crystal lattices, suggesting a mechanism by which distinctive ectodomain surfaces determine clustering, and thereby signaling, properties. © 2013 Nature America, Inc. All rights reserved.

Seiradake E, Schaupp A, del Toro Ruiz D, Kaufmann R, Mitakidis N, Harlos K, Aricescu AR, Klein R, Jones EY. 2013. Structurally encoded intraclass differences in EphA clusters drive distinct cell responses. Nat Struct Mol Biol, 20 (8), pp. 958-964. | Show Abstract | Read more

Functional outcomes of ephrin binding to Eph receptors (Ephs) range from cell repulsion to adhesion. Here we used cell collapse and stripe assays, showing contrasting effects of human ephrinA5 binding to EphA2 and EphA4. Despite equivalent ligand binding affinities, EphA4 triggered greater cell collapse, whereas EphA2-expressing cells adhered better to ephrinA5-coated surfaces. Chimeric receptors showed that the ectodomain is a major determinant of cell response. We report crystal structures of EphA4 ectodomain alone and in complexes with ephrinB3 and ephrinA5. These revealed closed clusters with a dimeric or circular arrangement in the crystal lattice, contrasting with extended arrays previously observed for EphA2 ectodomain. Localization microscopy showed that ligand-stimulated EphA4 induces smaller clusters than does EphA2. Mutant Ephs link these characteristics to interactions observed in the crystal lattices, suggesting a mechanism by which distinctive ectodomain surfaces determine clustering, and thereby signaling, properties.

Rat Genome Sequencing and Mapping Consortium, Baud A, Hermsen R, Guryev V, Stridh P, Graham D, McBride MW, Foroud T, Calderari S, Diez M et al. 2013. Combined sequence-based and genetic mapping analysis of complex traits in outbred rats. Nat Genet, 45 (7), pp. 767-775. | Show Abstract | Read more

Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.

Lu M, Breyssens H, Salter V, Zhong S, Hu Y, Baer C, Ratnayaka I, Sullivan A, Brown NR, Endicott J et al. 2013. Restoring p53 function in human melanoma cells by inhibiting MDM2 and cyclin B1/CDK1-phosphorylated nuclear iASPP. Cancer Cell, 23 (5), pp. 618-633. | Show Abstract | Read more

Nearly 90% of human melanomas contain inactivated wild-type p53, the underlying mechanisms for which are not fully understood. Here, we identify that cyclin B1/CDK1-phosphorylates iASPP, which leads to the inhibition of iASPP dimerization, promotion of iASPP monomer nuclear entry, and exposure of its p53 binding sites, leading to increased p53 inhibition. Nuclear iASPP is enriched in melanoma metastasis and associates with poor patient survival. Most wild-type p53-expressing melanoma cell lines coexpress high levels of phosphorylated nuclear iASPP, MDM2, and cyclin B1. Inhibition of MDM2 and iASPP phosphorylation with small molecules induced p53-dependent apoptosis and growth suppression. Concurrent p53 reactivation and BRAFV600E inhibition achieved additive suppression in vivo, presenting an alternative for melanoma therapy.

Cited:

26

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Siebold C, Jones EY. 2013. Structural insights into semaphorins and their receptors Seminars in Cell and Developmental Biology, 24 (3), pp. 139-145. | Show Abstract | Read more

Ten years ago nothing was known of the three-dimensional structure of members of the semaphorin family of cell guidance cues, nor of their major receptors, the plexins. The structural biology of this cell surface ligand-receptor system has now come of age. Detailed atomic level information is available on the architecture of semaphorin and plexin ectodomains and their recognition complexes. Similarly the structure of the plexin cytoplasmic region, and its interactions with members of the Rho family of small GTPases have been unveiled. These structural analyses, in combination with biochemical, biophysical and cellular studies, have progressed our understanding of this signalling system into the realm of molecular mechanism. © 2012 Elsevier Ltd.

Siebold C, Jones EY. 2013. Structural insights into semaphorins and their receptors. Semin Cell Dev Biol, 24 (3), pp. 139-145. | Show Abstract | Read more

Ten years ago nothing was known of the three-dimensional structure of members of the semaphorin family of cell guidance cues, nor of their major receptors, the plexins. The structural biology of this cell surface ligand-receptor system has now come of age. Detailed atomic level information is available on the architecture of semaphorin and plexin ectodomains and their recognition complexes. Similarly the structure of the plexin cytoplasmic region, and its interactions with members of the Rho family of small GTPases have been unveiled. These structural analyses, in combination with biochemical, biophysical and cellular studies, have progressed our understanding of this signalling system into the realm of molecular mechanism.

Stewart-Jones GB, Simpson P, van der Merwe PA, Easterbrook P, McMichael AJ, Rowland-Jones SL, Jones EY, Gillespie GM. 2012. Structural features underlying T-cell receptor sensitivity to concealed MHC class I micropolymorphisms. Proc Natl Acad Sci U S A, 109 (50), pp. E3483-E3492. | Show Abstract | Read more

Polymorphic differences distinguishing MHC class I subtypes often permit the presentation of shared epitopes in conformationally identical formats but can affect T-cell repertoire selection, differentially impacting autoimmune susceptibilities and viral clearance in vivo. The molecular mechanisms underlying this effect are not well understood. We performed structural, thermodynamic, and functional analyses of a conserved T-cell receptor (TCR) which is frequently expanded in response to a HIV-1 epitope when presented by HLA-B*5701 but is not selected by HLA-B*5703, which differs from HLA-B*5701 by two concealed polymorphisms. Our findings illustrate that although both HLA-B*57 subtypes display the epitope in structurally conserved formats, the impact of their polymorphic differences occurs directly as a consequence of TCR ligation, primarily because of peptide adjustments required for TCR binding, which involves the interplay of polymorphic residues and water molecules. These minor differences culminate in subtype-specific differential TCR-binding kinetics and cellular function. Our data demonstrate a potential mechanism whereby the most subtle MHC class I micropolymorphisms can influence TCR use and highlight their implications for disease outcomes.

Williams C, Hoppe HJ, Rezgui D, Strickland M, Forbes BE, Grutzner F, Frago S, Ellis RZ, Wattana-Amorn P, Prince SN et al. 2012. An exon splice enhancer primes IGF2:IGF2R binding site structure and function evolution. Science, 338 (6111), pp. 1209-1213. | Show Abstract | Read more

Placental development and genomic imprinting coevolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes IGF2 and IGF2R code for the growth promoter insulin-like growth factor 2 (IGF2) and its inhibitor, mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R), respectively. M6P/IGF2R of birds and fish do not recognize IGF2. In monotremes, which lack imprinting, IGF2 specifically bound M6P/IGF2R via a hydrophobic CD loop. We show that the DNA coding the CD loop in monotremes functions as an exon splice enhancer (ESE) and that structural evolution of binding site loops (AB, HI, FG) improved therian IGF2 affinity. We propose that ESE evolution led to the fortuitous acquisition of IGF2 binding by M6P/IGF2R that drew IGF2R into parental conflict; subsequent imprinting may then have accelerated affinity maturation.

Bowden TA, Baruah K, Coles CH, Harvey DJ, Yu X, Song BD, Stuart DI, Aricescu AR, Scanlan CN, Jones EY, Crispin M. 2012. Chemical and structural analysis of an antibody folding intermediate trapped during glycan biosynthesis. J Am Chem Soc, 134 (42), pp. 17554-17563. | Show Abstract | Read more

Human IgG Fc glycosylation modulates immunological effector functions such as antibody-dependent cellular cytotoxicity and phagocytosis. Engineering of Fc glycans therefore enables fine-tuning of the therapeutic properties of monoclonal antibodies. The N-linked glycans of Fc are typically complex-type, forming a network of noncovalent interactions along the protein surface of the Cγ2 domain. Here, we manipulate the mammalian glycan-processing pathway to trap IgG1 Fc at sequential stages of maturation, from oligomannose- to hybrid- to complex-type glycans, and show that the Fc is structurally stabilized following the transition of glycans from their hybrid- to complex-type state. X-ray crystallographic analysis of this hybrid-type intermediate reveals that N-linked glycans undergo conformational changes upon maturation, including a flip within the trimannosyl core. Our crystal structure of this intermediate reveals a molecular basis for antibody biogenesis and provides a template for the structure-guided engineering of the protein-glycan interface of therapeutic antibodies.

Jones EY, Yeates TO. 2012. Structure and function in complex macromolecular assemblies: some evolutionary themes. Curr Opin Struct Biol, 22 (2), pp. 197-199. | Read more

Janssen BJ, Malinauskas T, Weir GA, Cader MZ, Siebold C, Jones EY. 2012. Neuropilins lock secreted semaphorins onto plexins in a ternary signaling complex. Nat Struct Mol Biol, 19 (12), pp. 1293-1299. | Show Abstract | Read more

Co-receptors add complexity to cell-cell signaling systems. The secreted semaphorin 3s (Sema3s) require a co-receptor, neuropilin (Nrp), to signal through plexin As (PlxnAs) in functions ranging from axon guidance to bone homeostasis, but the role of the co-receptor is obscure. Here we present the low-resolution crystal structure of a mouse semaphorin-plexin-Nrp complex alongside unliganded component structures. Dimeric semaphorin, two copies of plexin and two copies of Nrp are arranged as a dimer of heterotrimers. In each heterotrimer subcomplex, semaphorin contacts plexin, similar to in co-receptor-independent signaling complexes. The Nrp1s cross brace the assembly, bridging between sema domains of the Sema3A and PlxnA2 subunits from the two heterotrimers. Biophysical and cellular analyses confirm that this Nrp binding mode stabilizes a canonical, but weakened, Sema3-PlxnA interaction, adding co-receptor control over the mechanism by which receptor dimerization and/or oligomerization triggers signaling.

Bourhis JM, Mariano N, Zhao Y, Harlos K, Exposito JY, Jones EY, Moali C, Aghajari N, Hulmes DJ. 2012. Structural basis of fibrillar collagen trimerization and related genetic disorders. Nat Struct Mol Biol, 19 (10), pp. 1031-1036. | Show Abstract | Read more

The C propeptides of fibrillar procollagens have crucial roles in tissue growth and repair by controlling both the intracellular assembly of procollagen molecules and the extracellular assembly of collagen fibrils. Mutations in C propeptides are associated with several, often lethal, genetic disorders affecting bone, cartilage, blood vessels and skin. Here we report the crystal structure of a C-propeptide domain from human procollagen III. It reveals an exquisite structural mechanism of chain recognition during intracellular trimerization of the procollagen molecule. It also gives insights into why some types of collagen consist of three identical polypeptide chains, whereas others do not. Finally, the data show striking correlations between the sites of numerous disease-related mutations in different C-propeptide domains and the degree of phenotype severity. The results have broad implications for understanding genetic disorders of connective tissues and designing new therapeutic strategies.

Seiradake E, Coles CH, Perestenko PV, Harlos K, McIlhinney RA, Aricescu AR, Jones EY. 2011. Structural basis for cell surface patterning through NetrinG-NGL interactions. EMBO J, 30 (21), pp. 4479-4488. | Show Abstract | Read more

Brain wiring depends on cells making highly localized and selective connections through surface protein-protein interactions, including those between NetrinGs and NetrinG ligands (NGLs). The NetrinGs are members of the structurally uncharacterized netrin family. We present a comprehensive crystallographic analysis comprising NetrinG1-NGL1 and NetrinG2-NGL2 complexes, unliganded NetrinG2 and NGL3. Cognate NetrinG-NGL interactions depend on three specificity-conferring NetrinG loops, clasped tightly by matching NGL surfaces. We engineered these NGL surfaces to implant custom-made affinities for NetrinG1 and NetrinG2. In a cellular patterning assay, we demonstrate that NetrinG-binding selectivity can direct the sorting of a mixed population of NGLs into discrete cell surface subdomains. These results provide a molecular model for selectivity-based patterning in a neuronal recognition system, dysregulation of which is associated with severe neuropsychological disorders.

Malinauskas T, Aricescu AR, Lu W, Siebold C, Jones EY. 2011. Modular mechanism of Wnt signaling inhibition by Wnt inhibitory factor 1. Nat Struct Mol Biol, 18 (8), pp. 886-893. | Show Abstract | Read more

Wnt morphogens control embryonic development and homeostasis in adult tissues. In vertebrates the N-terminal WIF domain (WIF-1(WD)) of Wnt inhibitory factor 1 (WIF-1) binds Wnt ligands. Our crystal structure of WIF-1(WD) reveals a previously unidentified binding site for phospholipid; two acyl chains extend deep into the domain, and the head group is exposed to the surface. Biophysical and cellular assays indicate that there is a WIF-1(WD) Wnt-binding surface proximal to the lipid head group but also implicate the five epidermal growth factor (EGF)-like domains (EGFs I-V) in Wnt binding. The six-domain WIF-1 crystal structure shows that EGFs I-V are wrapped back, interfacing with WIF-1(WD) at EGF III. EGFs II-V contain a heparan sulfate proteoglycan (HSPG)-binding site, consistent with conserved positively charged residues on EGF IV. This combination of HSPG- and Wnt-binding properties suggests a modular model for the localization of WIF-1 and for signal inhibition within morphogen gradients.

Zhao Y, Bishop B, Clay JE, Lu W, Jones M, Daenke S, Siebold C, Stuart DI, Jones EY, Aricescu AR. 2011. Automation of large scale transient protein expression in mammalian cells. J Struct Biol, 175 (2), pp. 209-215. | Show Abstract | Read more

Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI⁻ cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative.

McMahon RM, Friis L, Siebold C, Friese MA, Fugger L, Jones EY. 2011. Structure of HLA-A*0301 in complex with a peptide of proteolipid protein: insights into the role of HLA-A alleles in susceptibility to multiple sclerosis. Acta Crystallogr D Biol Crystallogr, 67 (Pt 5), pp. 447-454. | Show Abstract | Read more

The structure of the human major histocompatability (MHC) class I molecule HLA-A*0301 (HLA-A3) in complex with a nonameric peptide (KLIETYFSK) has been determined by X-ray crystallography to 2.7 Å resolution. HLA-A3 is a predisposing allele for multiple sclerosis (MS), an autoimmune disease of the central nervous system. The KLIETYFSK peptide is a naturally processed epitope of proteolipid protein, a myelin protein and candidate target for immune-mediated myelin destruction in MS. Comparison of the structure of HLA-A3 with that of HLA-A2, an MHC class I molecule which is protective against MS, indicates that both MHC class I molecules present very similar faces for T-cell receptor recognition whilst differing in the specificity of their peptide-binding grooves. These characteristics may underlie the opposing (predisposing versus protective) associations that they exhibit both in humans and in mouse models of MS-like disease. Furthermore, subtle alterations within the peptide-binding groove of HLA-A3 and other A3-like MHC class I molecules, members of the so-called A3 superfamily, may be sufficient to alter their presentation of autoantigen peptides such as KLIETYFSK. This in turn may modulate their contribution to the associated risk of autoimmune disease.

Bowden TA, Jones EY, Stuart DI. 2011. Cells under siege: viral glycoprotein interactions at the cell surface. J Struct Biol, 175 (2), pp. 120-126. | Show Abstract | Read more

As obligate parasites, viruses are required to enter and replicate within their host, a process which employs many of their proteins to hijack natural cellular processes. High resolution X-ray crystallographic analysis has proven to be an ideal method to visualize the mechanisms by which such virus-host interactions occur and has revealed the innovative capacity of viruses to adapt efficiently to their hosts. In this review, we draw upon recently elucidated paramyxovirus-, arenavirus-, and poxvirus-host protein complex crystal structures to reveal both the capacity of viruses to appropriate one component of a physiological protein-protein binding event (often modifying it to out-compete the host-protein), and the ability to utilize novel binding sites on host cell surface receptors. The structures discussed shed light on a number of biological processes ranging from viral entry to virulence and host antagonism. Drawn together they reveal the common strategies which viruses have evolved to interact with their natural host. The structures also support molecular level rationales for how viruses can be transmitted to unrelated organisms and thus pose severe health risks.

Bubeck D, Reijns MA, Graham SC, Astell KR, Jones EY, Jackson AP. 2011. PCNA directs type 2 RNase H activity on DNA replication and repair substrates. Nucleic Acids Res, 39 (9), pp. 3652-3666. | Show Abstract | Read more

Ribonuclease H2 is the major nuclear enzyme degrading cellular RNA/DNA hybrids in eukaryotes and the sole nuclease known to be able to hydrolyze ribonucleotides misincorporated during genomic replication. Mutation in RNASEH2 causes Aicardi-Goutières syndrome, an auto-inflammatory disorder that may arise from nucleic acid byproducts generated during DNA replication. Here, we report the crystal structures of Archaeoglobus fulgidus RNase HII in complex with PCNA, and human PCNA bound to a C-terminal peptide of RNASEH2B. In the archaeal structure, three binding modes are observed as the enzyme rotates about a flexible hinge while anchored to PCNA by its PIP-box motif. PCNA binding promotes RNase HII activity in a hinge-dependent manner. It enhances both cleavage of ribonucleotides misincorporated in DNA duplexes, and the comprehensive hydrolysis of RNA primers formed during Okazaki fragment maturation. In addition, PCNA imposes strand specificity on enzyme function, and by localizing RNase H2 and not RNase H1 to nuclear replication foci in vivo it ensures that RNase H2 is the dominant RNase H activity during nuclear replication. Our findings provide insights into how type 2 RNase H activity is directed during genome replication and repair, and suggest a mechanism by which RNase H2 may suppress generation of immunostimulatory nucleic acids.

Chen S, Bubeck D, MacDonald BT, Liang WX, Mao JH, Malinauskas T, Llorca O, Aricescu AR, Siebold C, He X, Jones EY. 2011. Structural and functional studies of LRP6 ectodomain reveal a platform for Wnt signaling. Dev Cell, 21 (5), pp. 848-861. | Show Abstract | Read more

LDL-receptor-related protein 6 (LRP6), alongside Frizzled receptors, transduces Wnt signaling across the plasma membrane. The LRP6 ectodomain comprises four tandem β-propeller-EGF-like domain (PE) pairs that harbor binding sites for Wnt morphogens and their antagonists including Dickkopf 1 (Dkk1). To understand how these multiple interactions are integrated, we combined crystallographic analysis of the third and fourth PE pairs with electron microscopy (EM) to determine the complete ectodomain structure. An extensive inter-pair interface, conserved for the first-to-second and third-to-fourth PE interactions, contributes to a compact platform-like architecture, which is disrupted by mutations implicated in developmental diseases. EM reconstruction of the LRP6 platform bound to chaperone Mesd exemplifies a binding mode spanning PE pairs. Cellular and binding assays identify overlapping Wnt3a- and Dkk1-binding surfaces on the third PE pair, consistent with steric competition, but also suggest a model in which the platform structure supports an interplay of ligands through multiple interaction sites.

Cited:

23

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Seiradake E, Coles CH, Perestenko PV, Harlos K, McIlhinney RAJ, Aricescu AR, Jones EY. 2011. Structural basis for cell surface patterning through NetrinG-NGL interactions EMBO Journal, 30 (21), pp. 4479-4488. | Show Abstract | Read more

Brain wiring depends on cells making highly localized and selective connections through surface protein-protein interactions, including those between NetrinGs and NetrinG ligands (NGLs). The NetrinGs are members of the structurally uncharacterized netrin family. We present a comprehensive crystallographic analysis comprising NetrinG1-NGL1 and NetrinG2-NGL2 complexes, unliganded NetrinG2 and NGL3. Cognate NetrinG-NGL interactions depend on three specificity-conferring NetrinG loops, clasped tightly by matching NGL surfaces. We engineered these NGL surfaces to implant custom-made affinities for NetrinG1 and NetrinG2. In a cellular patterning assay, we demonstrate that NetrinG-binding selectivity can direct the sorting of a mixed population of NGLs into discrete cell surface subdomains. These results provide a molecular model for selectivity-based patterning in a neuronal recognition system, dysregulation of which is associated with severe neuropsychological disorders. © 2011 European Molecular Biology Organization | All Rights Reserved.

Bell CH, Aricescu AR, Jones EY, Siebold C. 2011. A dual binding mode for RhoGTPases in plexin signalling. PLoS Biol, 9 (8), pp. e1001134. | Show Abstract | Read more

Plexins are cell surface receptors for the semaphorin family of cell guidance cues. The cytoplasmic region comprises a Ras GTPase-activating protein (GAP) domain and a RhoGTPase binding domain. Concomitant binding of extracellular semaphorin and intracellular RhoGTPase triggers GAP activity and signal transduction. The mechanism of this intricate regulation remains elusive. We present two crystal structures of the human Plexin-B1 cytoplasmic region in complex with a constitutively active RhoGTPase, Rac1. The structure of truncated Plexin-B1-Rac1 complex provides no mechanism for coupling RhoGTPase and Ras binding sites. On inclusion of the juxtamembrane helix, a trimeric structure of Plexin-B1-Rac1 complexes is stabilised by a second, novel, RhoGTPase binding site adjacent to the Ras site. Site-directed mutagenesis combined with cellular and biophysical assays demonstrate that this new binding site is essential for signalling. Our findings are consistent with a model in which extracellular and intracellular plexin clustering events combine into a single signalling output.

Cited:

21

Scopus

Bowden TA, Jones EY, Stuart DI. 2011. Cells under siege: Viral glycoprotein interactions at the cell surface Journal of Structural Biology, 175 (2), pp. 120-126. | Show Abstract | Read more

As obligate parasites, viruses are required to enter and replicate within their host, a process which employs many of their proteins to hijack natural cellular processes. High resolution X-ray crystallographic analysis has proven to be an ideal method to visualize the mechanisms by which such virus-host interactions occur and has revealed the innovative capacity of viruses to adapt efficiently to their hosts. In this review, we draw upon recently elucidated paramyxovirus-, arenavirus-, and poxvirus-host protein complex crystal structures to reveal both the capacity of viruses to appropriate one component of a physiological protein-protein binding event (often modifying it to out-compete the host-protein), and the ability to utilize novel binding sites on host cell surface receptors. The structures discussed shed light on a number of biological processes ranging from viral entry to virulence and host antagonism. Drawn together they reveal the common strategies which viruses have evolved to interact with their natural host. The structures also support molecular level rationales for how viruses can be transmitted to unrelated organisms and thus pose severe health risks. © 2011 Elsevier Inc.

Cited:

58

Scopus

Malinauskas T, Aricescu AR, Lu W, Siebold C, Jones EY. 2011. Modular mechanism of Wnt signaling inhibition by Wnt inhibitory factor 1 Nature Structural and Molecular Biology, 18 (8), pp. 886-893. | Show Abstract | Read more

Wnt morphogens control embryonic development and homeostasis in adult tissues. In vertebrates the N-terminal WIF domain (WIF-1 WD) of Wnt inhibitory factor 1 (WIF-1) binds Wnt ligands. Our crystal structure of WIF-1 WD reveals a previously unidentified binding site for phospholipid; two acyl chains extend deep into the domain, and the head group is exposed to the surface. Biophysical and cellular assays indicate that there is a WIF-1 WD Wnt-binding surface proximal to the lipid head group but also implicate the five epidermal growth factor (EGF)-like domains (EGFs I-V) in Wnt binding. The six-domain WIF-1 crystal structure shows that EGFs I-V are wrapped back, interfacing with WIF-1 WD at EGF III. EGFs II-V contain a heparan sulfate proteoglycan (HSPG)-binding site, consistent with conserved positively charged residues on EGF IV. This combination of HSPG-and Wnt-binding properties suggests a modular model for the localization of WIF-1 and for signal inhibition within morphogen gradients. © 2011 Nature America, Inc. All rights reserved.

Coles CH, Shen Y, Tenney AP, Siebold C, Sutton GC, Lu W, Gallagher JT, Jones EY, Flanagan JG, Aricescu AR. 2011. Proteoglycan-specific molecular switch for RPTPσ clustering and neuronal extension. Science, 332 (6028), pp. 484-488. | Show Abstract | Read more

Heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) regulate numerous cell surface signaling events, with typically opposite effects on cell function. CSPGs inhibit nerve regeneration through receptor protein tyrosine phosphatase sigma (RPTPσ). Here we report that RPTPσ acts bimodally in sensory neuron extension, mediating CSPG inhibition and HSPG growth promotion. Crystallographic analyses of a shared HSPG-CSPG binding site reveal a conformational plasticity that can accommodate diverse glycosaminoglycans with comparable affinities. Heparan sulfate and analogs induced RPTPσ ectodomain oligomerization in solution, which was inhibited by chondroitin sulfate. RPTPσ and HSPGs colocalize in puncta on sensory neurons in culture, whereas CSPGs occupy the extracellular matrix. These results lead to a model where proteoglycans can exert opposing effects on neuronal extension by competing to control the oligomerization of a common receptor.

Reijns MA, Bubeck D, Gibson LC, Graham SC, Baillie GS, Jones EY, Jackson AP. 2011. The structure of the human RNase H2 complex defines key interaction interfaces relevant to enzyme function and human disease. J Biol Chem, 286 (12), pp. 10530-10539. | Show Abstract | Read more

Ribonuclease H2 (RNase H2) is the major nuclear enzyme involved in the degradation of RNA/DNA hybrids and removal of ribonucleotides misincorporated in genomic DNA. Mutations in each of the three RNase H2 subunits have been implicated in a human auto-inflammatory disorder, Aicardi-Goutières Syndrome (AGS). To understand how mutations impact on RNase H2 function we determined the crystal structure of the human heterotrimer. In doing so, we correct several key regions of the previously reported murine RNase H2 atomic model and provide biochemical validation for our structural model. Our results provide new insights into how the subunits are arranged to form an enzymatically active complex. In particular, we establish that the RNASEH2A C terminus is a eukaryotic adaptation for binding the two accessory subunits, with residues within it required for enzymatic activity. This C-terminal extension interacts with the RNASEH2C C terminus and both are necessary to form a stable, enzymatically active heterotrimer. Disease mutations cluster at this interface between all three subunits, destabilizing the complex and/or impairing enzyme activity. Altogether, we locate 25 out of 29 residues mutated in AGS patients, establishing a firm basis for future investigations into disease pathogenesis and function of the RNase H2 enzyme.

McMichael AJ, Jones EY. 2010. Genetics. First-class control of HIV-1. Science, 330 (6010), pp. 1488-1490. | Show Abstract | Read more

Genome-wide association studies reveal amino acids of the major histocompatibility complex that associate with the rate of progression to AIDS.

Bowden TA, Crispin M, Jones EY, Stuart DI. 2010. Shared paramyxoviral glycoprotein architecture is adapted for diverse attachment strategies. Biochem Soc Trans, 38 (5), pp. 1349-1355. | Show Abstract | Read more

Members within the paramyxovirus subfamily Paramyxovirinae constitute a large number of highly virulent human and animal pathogens. The glycoproteins present on these viruses are responsible for mediating host cell attachment and fusion and are key targets for the design of antiviral entry inhibitors. In the present review, we discuss recent structural studies which have led to a better understanding of the various mechanisms by which different paramyxoviruses use their attachment glycoproteins to hijack specific protein and glycan cell-surface receptors to facilitate viral entry. It is observed that the paramyxovirus attachment glycoprotein consists of a conserved overall structure which includes an N-terminal six-bladed β-propeller domain which is responsible for cell receptor binding. Crystal structures of this domain from different biomedically important paramyxoviruses, including measles, Nipah, Hendra, Newcastle disease and parainfluenza viruses, alone and in complex with their functional cell-surface receptors, demonstrate three contrasting mechanisms of receptor engagement that paramyxoviruses have evolved to confer discreet protein- and glycan-receptor specificity. This structural information highlights the adaptability of the paramyxovirus attachment glycoprotein surface and the potential for the emergence of new and potentially harmful viruses in human hosts.

Janssen BJ, Robinson RA, Pérez-Brangulí F, Bell CH, Mitchell KJ, Siebold C, Jones EY. 2010. Structural basis of semaphorin-plexin signalling. Nature, 467 (7319), pp. 1118-1122. | Show Abstract | Read more

Cell-cell signalling of semaphorin ligands through interaction with plexin receptors is important for the homeostasis and morphogenesis of many tissues and is widely studied for its role in neural connectivity, cancer, cell migration and immune responses. SEMA4D and Sema6A exemplify two diverse vertebrate, membrane-spanning semaphorin classes (4 and 6) that are capable of direct signalling through members of the two largest plexin classes, B and A, respectively. In the absence of any structural information on the plexin ectodomain or its interaction with semaphorins the extracellular specificity and mechanism controlling plexin signalling has remained unresolved. Here we present crystal structures of cognate complexes of the semaphorin-binding regions of plexins B1 and A2 with semaphorin ectodomains (human PLXNB1(1-2)-SEMA4D(ecto) and murine PlxnA2(1-4)-Sema6A(ecto)), plus unliganded structures of PlxnA2(1-4) and Sema6A(ecto). These structures, together with biophysical and cellular assays of wild-type and mutant proteins, reveal that semaphorin dimers independently bind two plexin molecules and that signalling is critically dependent on the avidity of the resulting bivalent 2:2 complex (monomeric semaphorin binds plexin but fails to trigger signalling). In combination, our data favour a cell-cell signalling mechanism involving semaphorin-stabilized plexin dimerization, possibly followed by clustering, which is consistent with previous functional data. Furthermore, the shared generic architecture of the complexes, formed through conserved contacts of the amino-terminal seven-bladed β-propeller (sema) domains of both semaphorin and plexin, suggests that a common mode of interaction triggers all semaphorin-plexin based signalling, while distinct insertions within or between blades of the sema domains determine binding specificity.

van Boxel GI, Holmes S, Fugger L, Jones EY. 2010. An alternative conformation of the T-cell receptor alpha constant region. J Mol Biol, 400 (4), pp. 828-837. | Show Abstract | Read more

Alphabeta T-cell receptors (TcRs) play a central role in cellular immune response. They are members of the Ig superfamily, with extracellular regions of the alpha and beta chains each comprising a V-type domain and a C-type domain. We have determined the ectodomain structure of an alphabeta TcR, which recognizes the autoantigen myelin basic protein. The 2.0-A-resolution structure reveals canonical main-chain conformations for the V(alpha), V(beta), and C(beta) domains, but the C(alpha) domain exhibits a main-chain conformation remarkably different from those previously reported for TcR crystal structures. The global IgC-like fold is maintained, but a piston-like rearrangement between BC and DE beta-turns results in beta-strand slippage. This substantial conformational change may represent a signaling intermediate. Our structure is the first example for the Ig fold of the increasingly recognized concept of "metamorphic proteins."

Bowden TA, Crispin M, Harvey DJ, Jones EY, Stuart DI. 2010. Dimeric architecture of the Hendra virus attachment glycoprotein: evidence for a conserved mode of assembly. J Virol, 84 (12), pp. 6208-6217. | Show Abstract | Read more

Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed beta-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to alpha-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60 degrees ) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses.

Seiradake E, Harlos K, Sutton G, Aricescu AR, Jones EY. 2010. An extracellular steric seeding mechanism for Eph-ephrin signaling platform assembly. Nat Struct Mol Biol, 17 (4), pp. 398-402. | Show Abstract | Read more

Erythropoetin-producing hepatoma (Eph) receptors are cell-surface protein tyrosine kinases mediating cell-cell communication. Upon activation, they form signaling clusters. We report crystal structures of the full ectodomain of human EphA2 (eEphA2) both alone and in complex with the receptor-binding domain of the ligand ephrinA5 (ephrinA5 RBD). Unliganded eEphA2 forms linear arrays of staggered parallel receptors involving two patches of residues conserved across A-class Ephs. eEphA2-ephrinA5 RBD forms a more elaborate assembly, whose interfaces include the same conserved regions on eEphA2, but rearranged to accommodate ephrinA5 RBD. Cell-surface expression of mutant EphA2s showed that these interfaces are critical for localization at cell-cell contacts and activation-dependent degradation. Our results suggest a 'nucleation' mechanism whereby a limited number of ligand-receptor interactions 'seed' an arrangement of receptors which can propagate into extended signaling arrays.

Chen JL, Morgan AJ, Stewart-Jones G, Shepherd D, Bossi G, Wooldridge L, Hutchinson SL, Sewell AK, Griffiths GM, van der Merwe PA et al. 2010. Ca2+ release from the endoplasmic reticulum of NY-ESO-1-specific T cells is modulated by the affinity of TCR and by the use of the CD8 coreceptor. J Immunol, 184 (4), pp. 1829-1839. | Show Abstract | Read more

Although several cancer immunotherapy strategies are based on the use of analog peptides and on the modulation of the TCR affinity of adoptively transferred T cells, it remains unclear whether tumor-specific T cell activation by strong and weak TCR stimuli evoke different Ca(2+) signatures from the Ca(2+) intracellular stores and whether the amplitude of Ca(2+) release from the endoplasmic reticulum (ER) can be further modulated by coreceptor binding to peptide/MHC. In this study, we combined functional, structural, and kinetic measurements to correlate the intensity of Ca(2+) signals triggered by the stimulation of the 1G4 T cell clone specific to the tumor epitope NY-ESO-1(157-165). Two analogs of the NY-ESO-1(157-165) peptide, having similar affinity to HLA-A2 molecules, but a 6-fold difference in binding affinity for the 1G4 TCR, resulted in different Ca(2+) signals and T cell activation. 1G4 stimulation by the stronger stimulus emptied the ER of stored Ca(2+), even in the absence of CD8 binding, resulting in sustained Ca(2+) influx. In contrast, the weaker stimulus induced only partial emptying of stored Ca(2+), resulting in significantly diminished and oscillatory Ca(2+) signals, which were enhanced by CD8 binding. Our data define the range of TCR/peptide MHC affinities required to induce depletion of Ca(2+) from intracellular stores and provide insights into the ability of T cells to tailor the use of the CD8 coreceptor to enhance Ca(2+) release from the ER. This, in turn, modulates Ca(2+) influx from the extracellular environment, ultimately controlling T cell activation.

Macdonald IK, Harkiolaki M, Hunt L, Connelley T, Carroll AV, MacHugh ND, Graham SP, Jones EY, Morrison WI, Flower DR, Ellis SA. 2010. MHC class I bound to an immunodominant Theileria parva epitope demonstrates unconventional presentation to T cell receptors. PLoS Pathog, 6 (10), pp. e1001149. | Show Abstract | Read more

T cell receptor (TCR) recognition of peptide-MHC class I (pMHC) complexes is a crucial event in the adaptive immune response to pathogens. Peptide epitopes often display a strong dominance hierarchy, resulting in focusing of the response on a limited number of the most dominant epitopes. Such T cell responses may be additionally restricted by particular MHC alleles in preference to others. We have studied this poorly understood phenomenon using Theileria parva, a protozoan parasite that causes an often fatal lymphoproliferative disease in cattle. Despite its antigenic complexity, CD8+ T cell responses induced by infection with the parasite show profound immunodominance, as exemplified by the Tp1(214-224) epitope presented by the common and functionally important MHC class I allele N*01301. We present a high-resolution crystal structure of this pMHC complex, demonstrating that the peptide is presented in a distinctive raised conformation. Functional studies using CD8+ T cell clones show that this impacts significantly on TCR recognition. The unconventional structure is generated by a hydrophobic ridge within the MHC peptide binding groove, found in a set of cattle MHC alleles. Extremely rare in all other species, this feature is seen in a small group of mouse MHC class I molecules. The data generated in this analysis contribute to our understanding of the structural basis for T cell-dependent immune responses, providing insight into what determines a highly immunogenic p-MHC complex, and hence can be of value in prediction of antigenic epitopes and vaccine design.

Brown J, Jones EY, Forbes BE. 2009. Keeping IGF-II under control: lessons from the IGF-II-IGF2R crystal structure. Trends Biochem Sci, 34 (12), pp. 612-619. | Show Abstract | Read more

Insulin-like growth factor-II (IGF-II) is a key regulator of cell growth, survival, migration and differentiation. Its pivotal role in these processes requires tight regulation of both expression and activity. The type 1 IGF receptor tyrosine kinase (IGF-1R) mediates IGF-II actions, and a family of six high affinity IGF binding proteins (IGFBPs) regulates IGF-II circulating half-life and its availability to bind IGF-1R. In addition, the type 2 IGF receptor (IGF2R; also called the cation-independent mannose-6-phosphate receptor) modulates the circulating and tissue levels of IGF-II by targeting it to lysosomes for degradation. The recently elucidated crystal structure of IGF-II-IGF2R complex provides new insight into IGF-II regulation, and reveals a common binding surface on IGF-II for the regulatory proteins, IGF2R and the IGFBPs.

van Boxel GI, Stewart-Jones G, Holmes S, Sainsbury S, Shepherd D, Gillespie GM, Harlos K, Stuart DI, Owens R, Jones EY. 2009. Some lessons from the systematic production and structural analysis of soluble (alpha)(beta) T-cell receptors. J Immunol Methods, 350 (1-2), pp. 14-21. | Show Abstract | Read more

T-cell receptors (TCRs) are membrane proteins which recognize antigens with high specificity forming the basis of the cellular immune response. The study of these receptors has been limited by the challenges in expressing sufficient quantities of stable soluble protein. Here we report our systematic approach for generating soluble, (alpha)(beta)-TCRs, for X-ray crystallographic studies. By using small-scale expression screens, novel standardized quality control mechanisms and crystallization and imaging robots we were able to add significantly to the current TCR structural database. Our success in crystallizing both isolated TCRs and Major histocompatibility complex (MHC):TCR complexes has provided us with sufficient data to develop focused crystallization screens, which have proved generically useful for the crystallization of this family of proteins and complexes.

Bowden TA, Aricescu AR, Nettleship JE, Siebold C, Rahman-Huq N, Owens RJ, Stuart DI, Jones EY. 2009. Structural plasticity of eph receptor A4 facilitates cross-class ephrin signaling. Structure, 17 (10), pp. 1386-1397. | Show Abstract | Read more

The EphA4 tyrosine kinase cell surface receptor regulates an array of physiological processes and is the only currently known class A Eph receptor that binds both A and B class ephrins with high affinity. We have solved the crystal structure of the EphA4 ligand binding domain alone and in complex with (1) ephrinB2 and (2) ephrinA2. This set of structures shows that EphA4 has significant conformational plasticity in its ligand binding face. In vitro binding data demonstrate that it has a higher affinity for class A than class B ligands. Structural analyses, drawing on previously reported Eph receptor structures, show that EphA4 in isolation and in complex with ephrinA2 resembles other class A Eph receptors but on binding ephrinB2 assumes structural hallmarks of the class B Eph receptors. This interactive plasticity reveals EphA4 as a structural chameleon, able to adopt both A and B class Eph receptor conformations, and thus provides a molecular basis for EphA-type cross-class reactivity.

Clayton A, Siebold C, Gilbert RJ, Sutton GC, Harlos K, McIlhinney RA, Jones EY, Aricescu AR. 2009. Crystal structure of the GluR2 amino-terminal domain provides insights into the architecture and assembly of ionotropic glutamate receptors. J Mol Biol, 392 (5), pp. 1125-1132. | Show Abstract | Read more

Ionotropic glutamate receptors are functionally diverse but have a common architecture, including the 400-residue amino-terminal domain (ATD). We report a 1.8-A resolution crystal structure of human GluR2-ATD. This dimeric structure provides a mechanism for how the ATDs can drive receptor assembly and subtype-restricted composition. Lattice contacts in a 4.1-A resolution crystal form reveal a tetrameric (dimer-dimer) arrangement consistent with previous cellular and cryo-electron microscopic data for full-length AMPA receptors.

Crispin M, Chang VT, Harvey DJ, Dwek RA, Evans EJ, Stuart DI, Jones EY, Lord JM, Spooner RA, Davis SJ. 2009. A human embryonic kidney 293T cell line mutated at the Golgi alpha-mannosidase II locus. J Biol Chem, 284 (32), pp. 21684-21695. | Show Abstract | Read more

Disruption of Golgi alpha-mannosidase II activity can result in type II congenital dyserythropoietic anemia and induce lupus-like autoimmunity in mice. Here, we isolated a mutant human embryonic kidney (HEK) 293T cell line called Lec36, which displays sensitivity to ricin that lies between the parental HEK 293T cells, in which the secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which produce only oligomannose-type N-linked glycans. Stem cell marker 19A was transiently expressed in the HEK 293T Lec36 cells and in parental HEK 293T cells with and without the potent Golgi alpha-mannosidase II inhibitor, swainsonine. Negative ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, were dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi alpha-mannosidase II: a point mutation that mapped to the active site was found in one allele, and an in-frame deletion of 12 nucleotides was found in the other allele. Expression of the wild type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and as a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia.

Bowden TA, Crispin M, Graham SC, Harvey DJ, Grimes JM, Jones EY, Stuart DI. 2009. Unusual molecular architecture of the machupo virus attachment glycoprotein. J Virol, 83 (16), pp. 8259-8265. | Show Abstract | Read more

New World arenaviruses, which cause severe hemorrhagic fever, rely upon their envelope glycoproteins for attachment and fusion into their host cell. Here we present the crystal structure of the Machupo virus GP1 attachment glycoprotein, which is responsible for high-affinity binding at the cell surface to the transferrin receptor. This first structure of an arenavirus glycoprotein shows that GP1 consists of a novel alpha/beta fold. This provides a blueprint of the New World arenavirus attachment glycoproteins and reveals a new architecture of viral attachment, using a protein fold of unknown origins.

Bishop B, Aricescu AR, Harlos K, O'Callaghan CA, Jones EY, Siebold C. 2009. Structural insights into hedgehog ligand sequestration by the human hedgehog-interacting protein HHIP. Nat Struct Mol Biol, 16 (7), pp. 698-703. | Show Abstract | Read more

Hedgehog (Hh) morphogens have fundamental roles in development, whereas dysregulation of Hh signaling leads to disease. Multiple cell-surface receptors are responsible for transducing and/or regulating Hh signals. Among these, the Hedgehog-interacting protein (Hhip) is a highly conserved, vertebrate-specific inhibitor of Hh signaling. We have solved a series of crystal structures for the human HHIP ectodomain and Desert hedgehog (DHH) in isolation, as well as HHIP in complex with DHH (HHIP-DHH) and Sonic hedgehog (Shh) (HHIP-Shh), with and without Ca2+. The interaction determinants, confirmed by biophysical studies and mutagenesis, reveal previously uncharacterized and distinct functions for the Hh Zn2+ and Ca2+ binding sites--functions that may be common to all vertebrate Hh proteins. Zn2+ makes a key contribution to the Hh-HHIP interface, whereas Ca2+ is likely to prevent electrostatic repulsion between the two proteins, suggesting an important modulatory role. This interplay of several metal binding sites suggests a tuneable mechanism for regulation of Hh signaling.

Stewart-Jones G, Wadle A, Hombach A, Shenderov E, Held G, Fischer E, Kleber S, Nuber N, Stenner-Liewen F, Bauer S et al. 2009. Rational development of high-affinity T-cell receptor-like antibodies (vol 106, pg 5784, 2009) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 106 (26), pp. 10872-10872. | Read more

Harkiolaki M, Tsirka T, Lewitzky M, Simister PC, Joshi D, Bird LE, Jones EY, O'Reilly N, Feller SM. 2009. Distinct binding modes of two epitopes in Gab2 that interact with the SH3C domain of Grb2. Structure, 17 (6), pp. 809-822. | Show Abstract | Read more

Grb2 and Gab2 form a complex implicated in normal cell signaling and cancer development. Binding of the Grb2SH3C domain to Gab2 is essential for the interaction, but molecular details remained undefined. Using peptide arrays and isothermal titration calorimetry, two Grb2SH3C binding sites in Gab2 (Gab2a and Gab2b) were confirmed and characterized. Gab2a bears similarity to a p27Kip1 epitope that also binds Grb2SH3C. Crystal structures of both Gab2 epitopes complexed with Grb2SH3C reveal that Gab2b contains a 3(10) helix that positions the arginine and lysine of the core-binding motif RxxK in parallel orientation. In contrast, the Gab2a RxxK motif is embedded in a PPII helix with Arg and Lys in staggered orientation. A similar interaction mode is also present in a new complex of Mona/GadsSH3C with an RxxxxK epitope from the putative phosphatase HD-PTP. In summary, our study reveals interaction types of SH3 domains, highlighting their great versatility.

Tenzer S, Wee E, Burgevin A, Stewart-Jones G, Friis L, Lamberth K, Chang CH, Harndahl M, Weimershaus M, Gerstoft J et al. 2009. Antigen processing influences HIV-specific cytotoxic T lymphocyte immunodominance. Nat Immunol, 10 (6), pp. 636-646. | Show Abstract | Read more

Although cytotoxic T lymphocytes (CTLs) in people infected with human immunodeficiency virus type 1 can potentially target multiple virus epitopes, the same few are recognized repeatedly. We show here that CTL immunodominance in regions of the human immunodeficiency virus type 1 group-associated antigen proteins p17 and p24 correlated with epitope abundance, which was strongly influenced by proteasomal digestion profiles, affinity for the transporter protein TAP, and trimming mediated by the endoplasmatic reticulum aminopeptidase ERAAP, and was moderately influenced by HLA affinity. Structural and functional analyses demonstrated that proteasomal cleavage 'preferences' modulated the number and length of epitope-containing peptides, thereby affecting the response avidity and clonality of T cells. Cleavage patterns were affected by both flanking and intraepitope CTL-escape mutations. Our analyses show that antigen processing shapes CTL response hierarchies and that viral evolution modifies cleavage patterns and suggest strategies for in vitro vaccine optimization.

Walter BL, Armitage AE, Graham SC, de Oliveira T, Skinhøj P, Jones EY, Stuart DI, McMichael AJ, Chesebro B, Iversen AK. 2009. Functional characteristics of HIV-1 subtype C compatible with increased heterosexual transmissibility. AIDS, 23 (9), pp. 1047-1057. | Show Abstract | Read more

BACKGROUND: Despite the existence of over 50 subtypes and circulating recombinant forms of HIV-1, subtype C dominates the heterosexual pandemic causing approximately 56% of all infections. OBJECTIVE: To evaluate whether viral genetic factors may contribute to the observed subtype-C predominance. METHODS: Chimeric viruses were generated using V1-V3 envelope fragments from a subtype-A/C dually infected woman with preferential genital replication of subtype C. Viral adaptation, spread and cell fusion ability were evaluated in vitro using peripheral blood mononuclear cells and HeLa-CD4-CCR5 cell lines, sequencing and cloning. Structural modeling was performed using a crystal structure of gp120-CD4-X5. Phylogenetic analysis was done using subtype-A, subtype-B and subtype-C sequences from blood and cervix of 37 infected women and database sequences. RESULTS: We identified two envelope motifs, compact V1-V2 loops and V3-316T, which are found at high frequency throughout subtype-C evolution and affect gp120 interactions with CD4 and CCR5, respectively. When a V1-Delta5 deletion or V3-A316T was incorporated into subtype A, each increased viral fusion and spread several fold in peripheral blood mononuclear cell and cell lines with low CCR5 expression. Structural modeling suggested the formation of an additional hydrogen bond between V3 and CCR5. Moreover, we found preferential selection of HIV with 316T and/or extremely short V1-V2 loops in cervices of three women infected with subtypes A/C, B or C. CONCLUSION: As CD4-CCR5-T cells are key targets for genital HIV infection and cervical selection can favor compact V1-V2 loops and 316T, which increase viral infectivity, we propose that these conserved subtype-C motifs may contribute to transmission and spread of this subtype.

Crispin M, Bowden TA, Coles CH, Harlos K, Aricescu AR, Harvey DJ, Stuart DI, Jones EY. 2009. Carbohydrate and domain architecture of an immature antibody glycoform exhibiting enhanced effector functions. J Mol Biol, 387 (5), pp. 1061-1066. | Show Abstract | Read more

Antibodies contain a conserved glycosylation site that has emerged as a target for the modulation of antibody effector functions. The crystal structure of a biosynthetic intermediate of human IgG1, bearing immature oligomannose-type glycans and reported to display increased antibody-dependent cellular cytotoxicity, demonstrates that glycan engineering can bias the Fc to an open conformation primed for receptor binding.

Stewart-Jones G, Wadle A, Hombach A, Shenderov E, Held G, Fischer E, Kleber S, Nuber N, Stenner-Liewen F, Bauer S et al. 2009. Rational development of high-affinity T-cell receptor-like antibodies. Proc Natl Acad Sci U S A, 106 (14), pp. 5784-5788. | Show Abstract | Read more

T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1(157-165) peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW "peg" dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2-4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1(157-165) target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes.

Rezgui D, Williams C, Savage SA, Prince SN, Zaccheo OJ, Jones EY, Crump MP, Hassan AB. 2009. Structure and function of the human Gly1619Arg polymorphism of M6P/IGF2R domain 11 implicated in IGF2 dependent growth. J Mol Endocrinol, 42 (4), pp. 341-356. | Show Abstract | Read more

The mannose 6-phosphate/IGF 2 receptor (IGF2R) is comprised of 15 extra-cellular domains that bind IGF2 and mannose 6-phosphate ligands. IGF2R transports ligands from the Golgi to the pre-lysosomal compartment and thereafter to and from the cell surface. IGF2R regulates growth, placental development, tumour suppression and signalling. The ligand IGF2 is implicated in the growth phenotype, where IGF2R normally limits bioavailability, such that loss and gain of IGF2R results in increased and reduced growth respectively. The IGF2R exon 34 (5002A>G) polymorphism (rs629849) of the IGF2 specific binding domain has been correlated with impaired childhood growth (A/A homozygotes). We evaluated the function of the Gly1619Arg non-synonymous amino acid modification of domain 11. NMR and X-ray crystallography structures located 1619 remote from the ligand binding region of domain 11. Arg1619 was located close to the fibronectin type II (FnII) domain of domain 13, previously implicated as a modifier of IGF2 ligand binding through indirect interaction with the AB loop of the binding cleft. However, comparison of binding kinetics of IGF2R, Gly1619 and Arg1619 to either IGF2 or mannose 6-phosphate revealed no differences in 'on' and 'off' rates. Quantitative PCR, (35)S pulse chase and flow cytometry failed to demonstrate altered gene expression, protein half-life and cell membrane distribution, suggesting the polymorphism had no direct effect on receptor function. Intronic polymorphisms were identified which may be in linkage disequilibrium with rs629849 in certain populations. Other potential IGF2R polymorphisms may account for the correlation with childhood growth, warranting further functional evaluation.

Harkiolaki M, Holmes SL, Svendsen P, Gregersen JW, Jensen LT, McMahon R, Friese MA, van Boxel G, Etzensperger R, Tzartos JS et al. 2009. T cell-mediated autoimmune disease due to low-affinity crossreactivity to common microbial peptides. Immunity, 30 (3), pp. 348-357. | Show Abstract | Read more

Environmental factors account for 75% of the risk of developing multiple sclerosis (MS). Numerous infections have been suspected as environmental disease triggers, but none of them has consistently been incriminated, and it is unclear how so many different infections may play a role. We show that a microbial peptide, common to several major classes of bacteria, can induce MS-like disease in humanized mice by crossreacting with a T cell receptor (TCR) that also recognizes a peptide from myelin basic protein, a candidate MS autoantigen. Structural analysis demonstrates this crossreactivity is due to structural mimicry of a binding hotspot shared by self and microbial antigens, rather than to degenerate TCR recognition. Biophysical studies reveal that the autoreactive TCR binding affinity is markedly lower for the microbial (mimicry) peptide than for the autoantigenic peptide. Thus, these data suggest a possible explanation for the difficulty in incriminating individual infections in the development of MS.

Tenzer S, Wee E, Burgevin A, Stewart-Jones G, Friis L, Lamberth K, Chang C, Harndahl M, Weimershaus M, Gerstoft J et al. 2009. Antigen processing influences HIV-specific cytotoxic T lymphocyte immunodominance RETROVIROLOGY, 6 (Suppl 3), pp. P252-P252. | Read more

Brown J, Jones EY, Forbes BE. 2009. Interactions of IGF-II with the IGF2R/cation-independent mannose-6-phosphate receptor mechanism and biological outcomes. Vitam Horm, 80 pp. 699-719. | Show Abstract | Read more

The cation-independent mannose-6-phosphate/insulin-like growth factor-II receptor (IGF2R) is a membrane-bound glycoprotein consisting of 15 homologous extracellular repeat domains. The major function of this receptor is trafficking of lysosomal enzymes from the trans-Golgi network to the endosomes and their subsequent transfer to lysosomes. The IGF2R also plays a major role in binding and regulating the circulating and tissue levels of IGF-II. As this ligand is important for cell growth, survival, and migration, the maintenance of correct IGF-II levels influences its actions in normal growth and development. Deregulation of IGF2R expression has therefore been associated with growth related disease and cancer. This review highlights recent advances in understanding the IGF2R structure and mechanism of interaction with its ligands, in particular IGF-II. Recent mutagenesis studies combined with the crystal structure of domains 11-14 in complex with IGF-II have mapped the sites of interaction and explain how the IGF2R specificity for IGF-II is achieved. The role of domain 13 in high-affinity IGF-II binding is also revealed. Characterization of ligand:IGF2R interactions is vital for the understanding of the mechanism of IGF2R actions and will allow the development of specific cancer therapies in the future.

Stewart-Jones G, Wadle A, Hombach A, Shenderov E, Held G, Fischer E, Kleber S, Nuber N, Stenner-Liewen F, Bauer S et al. 2009. Correction for Stewart-Jones et al., Rational development of high-affinity T-cell receptor-like antibodies Proceedings of the National Academy of Sciences, 106 (26), pp. 10872-10872. | Read more

Bowden TA, Crispin M, Harvey DJ, Aricescu AR, Grimes JM, Jones EY, Stuart DI. 2008. Crystal structure and carbohydrate analysis of Nipah virus attachment glycoprotein: a template for antiviral and vaccine design. J Virol, 82 (23), pp. 11628-11636. | Show Abstract | Read more

Two members of the paramyxovirus family, Nipah virus (NiV) and Hendra virus (HeV), are recent additions to a growing number of agents of emergent diseases which use bats as a natural host. Identification of ephrin-B2 and ephrin-B3 as cellular receptors for these viruses has enabled the development of immunotherapeutic reagents which prevent virus attachment and subsequent fusion. Here we present the structural analysis of the protein and carbohydrate components of the unbound viral attachment glycoprotein of NiV glycoprotein (NiV-G) at a 2.2-A resolution. Comparison with its ephrin-B2-bound form reveals that conformational changes within the envelope glycoprotein are required to achieve viral attachment. Structural differences are particularly pronounced in the 579-590 loop, a major component of the ephrin binding surface. In addition, the 236-245 loop is rather disordered in the unbound structure. We extend our structural characterization of NiV-G with mass spectrometric analysis of the carbohydrate moieties. We demonstrate that NiV-G is largely devoid of the oligomannose-type glycans that in viruses such as human immunodeficiency virus type 1 and Ebola virus influence viral tropism and the host immune response. Nevertheless, we find putative ligands for the endothelial cell lectin, LSECtin. Finally, by mapping structural conservation and glycosylation site positions from other members of the paramyxovirus family, we suggest the molecular surface involved in oligomerization. These results suggest possible pathways of virus-host interaction and strategies for the optimization of recombinant vaccines.

Etzensperger R, McMahon RM, Jones EY, Fugger L. 2008. Dissection of the multiple sclerosis associated DR2 haplotype. J Autoimmun, 31 (3), pp. 201-207. | Show Abstract | Read more

Epidemiological and genetic data have consistently identified associations with HLA class II alleles in many autoimmune diseases. In multiple sclerosis (MS), an autoimmune disease targeting central nervous system (CNS) myelin, the DR2 haplotype (DRB1*1501, DRB5*0101 and DQB1*0602) remains the strongest identified genetic risk factor in Caucasians. However, it is hard to tease apart the precise contributions of its constituent individual alleles and their modes of action remain poorly understood, due in part to the strong linkage disequilibrium in this region. Recent work in humanized mice indicates functional epistatic interactions whereby DRB5*0101 directly modulates the severity of the ensuing disease through activation-induced cell death (AICD) of encephalitogenic T cells which are restricted by DRB1*1501. Complementary structural studies help to explain how these alleles may facilitate thymic escape of autoreactive T cells and contribute to peripheral T cell activation via suboptimal binding interactions and mechanisms of molecular mimicry. Here we discuss the emerging role of the constituent alleles of the DR2 haplotype and our ongoing efforts to uncover the mechanisms by which they influence MS pathogenesis.

Friese MA, Jakobsen KB, Friis L, Etzensperger R, Craner MJ, McMahon RM, Jensen LT, Huygelen V, Jones EY, Bell JI, Fugger L. 2008. Opposing effects of HLA class I molecules in tuning autoreactive CD8+ T cells in multiple sclerosis. Nat Med, 14 (11), pp. 1227-1235. | Show Abstract | Read more

The major known genetic risk factors in multiple sclerosis reside in the major histocompatibility complex (MHC) region. Although there is strong evidence implicating MHC class II alleles and CD4(+) T cells in multiple sclerosis pathogenesis, possible contributions from MHC class I genes and CD8(+) T cells are controversial. We have generated humanized mice expressing the multiple sclerosis-associated MHC class I alleles HLA-A(*)0301 (encoding human leukocyte antigen-A3 (HLA-A3)) and HLA-A(*)0201 (encoding HLA-A2) and a myelin-specific autoreactive T cell receptor (TCR) derived from a CD8(+) T cell clone from an individual with multiple sclerosis to study mechanisms of disease susceptibility. We demonstrate roles for HLA-A3-restricted CD8(+) T cells in induction of multiple sclerosis-like disease and for CD4(+) T cells in its progression, and we also define a possible mechanism for HLA-A(*)0201-mediated protection. To our knowledge, these data provide the first direct evidence incriminating MHC class I genes and CD8(+) T cells in the pathogenesis of human multiple sclerosis and reveal a network of MHC interactions that shape the risk of multiple sclerosis.

Bowden TA, Aricescu AR, Gilbert RJ, Grimes JM, Jones EY, Stuart DI. 2008. Structural basis of Nipah and Hendra virus attachment to their cell-surface receptor ephrin-B2. Nat Struct Mol Biol, 15 (6), pp. 567-572. | Show Abstract | Read more

Nipah and Hendra viruses are emergent paramyxoviruses, causing disease characterized by rapid onset and high mortality rates, resulting in their classification as Biosafety Level 4 pathogens. Their attachment glycoproteins are essential for the recognition of the cell-surface receptors ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3). Here we report crystal structures of both Nipah and Hendra attachment glycoproteins in complex with human EFNB2. In contrast to previously solved paramyxovirus attachment complexes, which are mediated by sialic acid interactions, the Nipah and Hendra complexes are maintained by an extensive protein-protein interface, including a crucial phenylalanine side chain on EFNB2 that fits snugly into a hydrophobic pocket on the viral protein. By analogy with the development of antivirals against sialic acid binding viruses, these results provide a structural template to target antiviral inhibition of protein-protein interactions.

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Silk JD, Salio M, Reddy BG, Shepherd D, Gileadi U, Brown J, Masri SH, Polzella P, Ritter G, Besra GS et al. 2008. Nonglycosidic CD1d lipid ligands activate human and murine invariant NKT cells JOURNAL OF IMMUNOLOGY, 180 (10), pp. 6452-6456.

Silk JD, Salio M, Reddy BG, Shepherd D, Gileadi U, Brown J, Masri SH, Polzella P, Ritter G, Besra GS et al. 2008. Cutting edge: nonglycosidic CD1d lipid ligands activate human and murine invariant NKT cells. J Immunol, 180 (10), pp. 6452-6456. | Show Abstract

Invariant NKT cells (iNKT cells) recognize CD1d/glycolipid complexes. We demonstrate that the nonglycosidic compound threitolceramide efficiently activates iNKT cells, resulting in dendritic cell (DC) maturation and the priming of Ag-specific T and B cells. Threitolceramide-pulsed DCs are more resistant to iNKT cell-dependent lysis than alpha-galactosylceramide-pulsed DCs due to the weaker affinity of the human iNKT TCR for CD1d/ threitolceramide than CD1d/alpha-galactosylceramide complexes. iNKT cells stimulated with threitolceramide also recover more quickly from activation-induced anergy. Kinetic and functional experiments showed that shortening or lengthening the threitol moiety by one hydroxymethylene group modulates ligand recognition, as human and murine iNKT cells recognize glycerolceramide and arabinitolceramide differentially. Our data broaden the range of potential iNKT cell agonists. The ability of these compounds to assist the priming of Ag-specific immune responses while minimizing iNKT cell-dependent DC lysis makes them attractive adjuvants for vaccination strategies.

Aricescu AR, Siebold C, Jones EY. 2008. Receptor protein tyrosine phosphatase micro: measuring where to stick. Biochem Soc Trans, 36 (Pt 2), pp. 167-172. | Show Abstract | Read more

We review here recent results on the structure and function of a receptor protein tyrosine phosphatase, RPTPmicro. In addition to their intercellular catalytic domains which bear the phosphatase activity, the RPTPs are cell-surface-receptor-type molecules and in many cases have large extracellular regions. What role can these extracellular regions play in function? For RPTPmicro, the extracellular region is known to mediate homophilic adhesion. Sequence analysis indicates that it comprises six domains: an N-terminal MAM (meprin/A5/micro), one immunoglobulin-like domain and four fibronectin type III (FN) repeats. We have determined the crystal structure of the entire extracellular region for RPTPmicro in the form of a functional adhesion dimer. The physical characteristics and dimensions of the adhesion dimer suggest a mechanism by which the location of this phosphatase can be influenced by cell-cell spacings.

Tabernero L, Aricescu AR, Jones EY, Szedlacsek SE. 2008. Protein tyrosine phosphatases: structure-function relationships. FEBS J, 275 (5), pp. 867-882. | Show Abstract | Read more

Structural analysis of protein tyrosine phosphatases (PTPs) has expanded considerably in the last several years, producing more than 200 structures in this class of enzymes (from 35 different proteins and their complexes with ligands). The small-medium size of the catalytic domain of approximately 280 residues plus a very compact fold makes it amenable to cloning and overexpression in bacterial systems thus facilitating crystallographic analysis. The low molecular weight PTPs being even smaller, approximately 150 residues, are also perfect targets for NMR analysis. The availability of different structures and complexes of PTPs with substrates and inhibitors has provided a wealth of information with profound effects in the way we understand their biological functions. Developments in mammalian expression technology recently led to the first crystal structure of a receptor-like PTP extracellular region. Altogether, the PTP structural work significantly advanced our knowledge regarding the architecture, regulation and substrate specificity of these enzymes. In this review, we compile the most prominent structural traits that characterize PTPs and their complexes with ligands. We discuss how the data can be used to design further functional experiments and as a basis for drug design given that many PTPs are now considered strategic therapeutic targets for human diseases such as diabetes and cancer.

Robinson RA, Lu X, Jones EY, Siebold C. 2008. Biochemical and structural studies of ASPP proteins reveal differential binding to p53, p63, and p73. Structure, 16 (2), pp. 259-268. | Show Abstract | Read more

ASPP1 and ASPP2 are activators of p53-dependent apoptosis, whereas iASPP is an inhibitor of p53. Binding assays showed differential binding for C-terminal domains of iASPP and ASPP2 to the core domains of p53 family members p53, p63, and p73. We also determined a high-resolution crystal structure for the C terminus of iASPP, comprised of four ankyrin repeats and an SH3 domain. The crystal lattice revealed an interaction between eight sequential residues in one iASPP molecule and the p53-binding site of a neighboring molecule. ITC confirmed that a peptide corresponding to the crystallographic interaction shows specific binding to iASPP. The contributions of ankyrin repeat residues, in addition to those of the SH3 domain, generate distinctive architecture at the p53-binding site suitable for inhibition by small molecules. These results suggest that the binding properties of iASPP render it a target for antitumor therapeutics and provide a peptide-based template for compound design.

Ishizuka J, Stewart-Jones GB, van der Merwe A, Bell JI, McMichael AJ, Jones EY. 2008. The structural dynamics and energetics of an immunodominant T cell receptor are programmed by its Vbeta domain. Immunity, 28 (2), pp. 171-182. | Show Abstract | Read more

Immunodominant and public T cell receptor (TCR) usage is relatively common in many viral diseases yet surprising in the context of the large naive TCR repertoire. We examined the highly conserved Vbeta17:Valpha10.2 JM22 T cell response to the influenza matrix peptide (58-66)-HLA-A*0201 (HLA-A2-flu) through extensive kinetic, thermodynamic, and structural analyses. We found several conformational adjustments that accompany JM22-HLA-A2-flu binding and identified a binding "hotspot" within the Vbeta domain of the TCR. Within this hotspot, key germline-encoded CDR1 and CDR2 loop residues and a crucial but commonly coded residue in the hypervariable region of CDR3 provide the basis for the substantial bias in the selection of the germline-encoded Vbeta17 domain. The chances of having a substantial number of T cells in the naive repertoire that have HLA-A2-flu-specific Vbeta17 receptors may consequently be relatively high, thus explaining the immunodominant usage of this clonotype.

Brown J, Delaine C, Zaccheo OJ, Siebold C, Gilbert RJ, van Boxel G, Denley A, Wallace JC, Hassan AB, Forbes BE, Jones EY. 2008. Structure and functional analysis of the IGF-II/IGF2R interaction. EMBO J, 27 (1), pp. 265-276. | Show Abstract | Read more

Embryonic development and normal growth require exquisite control of insulin-like growth factors (IGFs). In mammals the extracellular region of the cation-independent mannose-6-phosphate receptor has gained an IGF-II-binding function and is termed type II IGF receptor (IGF2R). IGF2R sequesters IGF-II; imbalances occur in cancers and IGF2R is implicated in tumour suppression. We report crystal structures of IGF2R domains 11-12, 11-12-13-14 and domains 11-12-13/IGF-II complex. A distinctive juxtaposition of these domains provides the IGF-II-binding unit, with domain 11 directly interacting with IGF-II and domain 13 modulating binding site flexibility. Our complex shows that Phe19 and Leu53 of IGF-II lock into a hydrophobic pocket unique to domain 11 of mammalian IGF2Rs. Mutagenesis analyses confirm this IGF-II 'binding-hotspot', revealing that IGF-binding proteins and IGF2R have converged on the same high-affinity site.

Silk JD, Salio M, Brown J, Jones EY, Cerundolo V. 2008. Structural and functional aspects of lipid binding by CD1 molecules. Annu Rev Cell Dev Biol, 24 (1), pp. 369-395. | Show Abstract | Read more

Over the past ten years, investigators have shown that T lymphocytes can recognize not only peptides in the context of MHC class I and class II molecules but also foreign and self-lipids in association with the nonclassical MHC class I molecules the CD1 proteins. We describe the events that have led to the discovery of the role of CD1 molecules, their pattern of intracellular trafficking, and their ability to sample different intracellular compartments for self- and foreign lipids. Structural and functional aspects of lipid presentation by CD1 molecules are presented in the context of the function of CD1-restricted T cells in antimicrobial responses, antitumor immunity, and the regulation of the tolerance and autoimmunity immunoregulatory axis. Particular emphasis is on invariant NKT (iNKT) cells and their ability to modulate innate and adaptive immune responses.

Koch M, Camp S, Collen T, Avila D, Salomonsen J, Wallny HJ, van Hateren A, Hunt L, Jacob JP, Johnston F et al. 2007. Structures of an MHC class I molecule from B21 chickens illustrate promiscuous peptide binding. Immunity, 27 (6), pp. 885-899. | Show Abstract | Read more

Little is known about the structure of major histocompatibility complex (MHC) molecules outside of mammals. Only one class I molecule in the chicken MHC is highly expressed, leading to strong genetic associations with infectious pathogens. Here, we report two structures of the MHC class I molecule BF2*2101 from the B21 haplotype, which is known to confer resistance to Marek's disease caused by an oncogenic herpesvirus. The binding groove has an unusually large central cavity, which confers substantial conformational flexibility to the crucial residue Arg9, allowing remodeling of key peptide-binding sites. The coupled variation of anchor residues from the peptide, utilizing a charge-transfer system unprecedented in MHC molecules, allows peptides with conspicuously different sequences to be bound. This promiscuous binding extends our understanding of ways in which MHC class I molecules can present peptides to the immune system and might explain the resistance of the B21 haplotype to Marek's disease.

Stronge VS, Salio M, Jones EY, Cerundolo V. 2007. A closer look at CD1d molecules: new horizons in studying NKT cells. Trends Immunol, 28 (10), pp. 455-462. | Show Abstract | Read more

Recent findings have highlighted the ability of invariant natural killer T (iNKT) cells to recognize microbe-derived glycolipids and have demonstrated the role of these cells in several disease states, from autoimmune disease to cancer. It has also become clear that iNKT cells can rapidly mature dendritic cells and licence them to prime antigen-specific T- and B-cell responses. The use of CD1d tetramers to monitor iNKT cell frequency and phenotype has moved the field forward at a fast pace. To harness iNKT cells for therapeutic purposes and to understand their role in vivo, it is essential to characterize the molecular events that contribute to iNKT cell activation. Here we review new reagents and novel protocols that are facilitating a closer look at lipid presentation by CD1d molecules and their recognition by iNKT cells.

Aricescu AR, Jones EY. 2007. Immunoglobulin superfamily cell adhesion molecules: zippers and signals. Curr Opin Cell Biol, 19 (5), pp. 543-550. | Show Abstract | Read more

The latest structural studies of immunoglobulin superfamily cell adhesion molecules are driving a shift in perspective; increasingly the view is not focused solely on the individual molecule but rather is on the molecular assembly. Two common themes are emerging, revealing mechanisms for ectodomain-dependent regulation of cell surface receptors' signalling abilities. The first is the propensity of many such molecules to arrange in zipper-type or array-type assemblies driven by a network of highly specific cis and trans interactions. The second is the use of the extracellular dimensions of a molecule or adhesion complex as properties which, in combination with characteristic intercellular spacings, can determine the co-localisation or exclusion of particular protein populations at cell interfaces and junctions.

Jones EY, Salio M, Cerundolo V. 2007. T cell receptors get back to basics. Nat Immunol, 8 (10), pp. 1033-1035. | Read more

Aricescu AR, Siebold C, Choudhuri K, Chang VT, Lu W, Davis SJ, van der Merwe PA, Jones EY. 2007. Structure of a tyrosine phosphatase adhesive interaction reveals a spacer-clamp mechanism. Science, 317 (5842), pp. 1217-1220. | Show Abstract | Read more

Cell-cell contacts are fundamental to multicellular organisms and are subject to exquisite levels of control. Human RPTPmu is a type IIB receptor protein tyrosine phosphatase that both forms an adhesive contact itself and is involved in regulating adhesion by dephosphorylating components of cadherin-catenin complexes. Here we describe a 3.1 angstrom crystal structure of the RPTPmu ectodomain that forms a homophilic trans (antiparallel) dimer with an extended and rigid architecture, matching the dimensions of adherens junctions. Cell surface expression of deletion constructs induces intercellular spacings that correlate with the ectodomain length. These data suggest that the RPTPmu ectodomain acts as a distance gauge and plays a key regulatory function, locking the phosphatase to its appropriate functional location.

Delaine C, Alvino CL, McNeil KA, Mulhern TD, Gauguin L, De Meyts P, Jones EY, Brown J, Wallace JC, Forbes BE. 2007. A novel binding site for the human insulin-like growth factor-II (IGF-II)/mannose 6-phosphate receptor on IGF-II. J Biol Chem, 282 (26), pp. 18886-18894. | Show Abstract | Read more

The mammalian insulin-like growth factor (IGF)-II/cation-independent mannose 6-phosphate receptor (IGF2R) binds IGF-II with high affinity. By targeting IGF-II to lysosomal degradation, it plays a role in the maintenance of correct IGF-II levels in the circulation and in target tissues. Loss of IGF2R function is associated with tumor progression; therefore, the IGF2R is often referred to as a tumor suppressor. The interaction between IGF2R and IGF-II involves domains 11 and 13 of the 15 extracellular domains of the receptor. Recently, a hydrophobic binding region was identified on domain 11 of the IGF2R. In contrast, relatively little is known about the residues of IGF-II that are involved in IGF2R binding and the determinants of IGF2R specificity for IGF-II over the structurally related IGF-I. Using a series of novel IGF-II analogues and surface plasmon resonance assays, this study revealed a novel binding surface on IGF-II critical for IGF2R binding. The hydrophobic residues Phe(19) and Leu(53) are critical for IGF2R binding, as are residues Thr(16) and Asp(52). Furthermore, Thr(16) was identified as playing a major role in determining why IGF-II, but not IGF-I, binds with high affinity to the IGF2R.

Brown J, O'Callaghan CA, Marshall AS, Gilbert RJ, Siebold C, Gordon S, Brown GD, Jones EY. 2007. Structure of the fungal beta-glucan-binding immune receptor dectin-1: implications for function. Protein Sci, 16 (6), pp. 1042-1052. | Show Abstract | Read more

The murine molecule dectin-1 (known as the beta-glucan receptor in humans) is an immune cell surface receptor implicated in the immunological defense against fungal pathogens. Sequence analysis has indicated that the dectin-1 extracellular domain is a C-type lectin-like domain, and functional studies have established that it binds fungal beta-glucans. We report several dectin-1 crystal structures, including a high-resolution structure and a 2.8 angstroms resolution structure in which a short soaked natural beta-glucan is trapped in the crystal lattice. In vitro characterization of dectin-1 in the presence of its natural ligand indicates higher-order complex formation between dectin-1 and beta-glucans. These combined structural and biophysical data considerably extend the current knowledge of dectin-1 structure and function, and suggest potential mechanisms of defense against fungal pathogens.

Crispin M, Aricescu AR, Chang VT, Jones EY, Stuart DI, Dwek RA, Davis SJ, Harvey DJ. 2007. Disruption of alpha-mannosidase processing induces non-canonical hybrid-type glycosylation. FEBS Lett, 581 (10), pp. 1963-1968. | Show Abstract | Read more

Golgi alpha-mannosidase II is essential for the efficient formation of complex-type glycosylation. Here, we demonstrate that the disruption of Golgi alpha-mannosidase II activity by swainsonine in human embryonic kidney cells is capable of inducing a novel class of hybrid-type glycosylation containing a partially processed mannose moiety. The discovery of 'Man(6)-based' hybrid-type glycans reveals a broader in vivo specificity of N-acetylglucosaminyltransferase I, further defines the arm-specific tolerance of core alpha1-6 fucosyltransferase to terminal alpha1-2 mannose residues, and suggests that disruption of Golgi alpha-mannosidase II activity is capable of inducing potentially 'non-self' structures.

McCarthy C, Shepherd D, Fleire S, Stronge VS, Koch M, Illarionov PA, Bossi G, Salio M, Denkberg G, Reddington F et al. 2007. The length of lipids bound to human CD1d molecules modulates the affinity of NKT cell TCR and the threshold of NKT cell activation. J Exp Med, 204 (5), pp. 1131-1144. | Show Abstract | Read more

CD1d-restricted lymphocytes recognize a broad lipid range. However, how CD1d-restricted lymphocytes translate T cell receptor (TCR) recognition of lipids with similar group heads into distinct biological responses remains unclear. Using a soluble invariant NKT (iNKT) TCR and a newly engineered antibody specific for alpha-galactosylceramide (alpha-GalCer)-human CD1d (hCD1d) complexes, we measured the affinity of binding of iNKT TCR to hCD1d molecules loaded with a panel of alpha-GalCer analogues and assessed the rate of dissociation of alpha-GalCer and alpha-GalCer analogues from hCD1d molecules. We extended this analysis by studying iNKT cell synapse formation and iNKT cell activation by the same panel of alpha-GalCer analogues. Our results indicate the unique role of the lipid chain occupying the hCD1d F' channel in modulating TCR binding affinity to hCD1d-lipid complexes, the formation of stable immunological synapse, and cell activation. These data are consistent with previously described conformational changes between empty and loaded hCD1d molecules (Koch, M., V.S. Stronge, D. Shepherd, S.D. Gadola, B. Mathew, G. Ritter, A.R. Fersht, G.S. Besra, R.R. Schmidt, E.Y. Jones, and V. Cerundolo. 2005. Nat. Immunol 6:819-826), suggesting that incomplete occupation of the hCD1d F' channel results in conformational differences at the TCR recognition surface. This indirect effect provides a general mechanism by which lipid-specific lymphocytes are capable of recognizing both the group head and the length of lipid antigens, ensuring greater specificity of antigen recognition.

Crispin M, Stuart DI, Jones EY. 2007. Building meaningful models of glycoproteins. Nat Struct Mol Biol, 14 (5), pp. 354. | Read more

Chotiyarnwong P, Stewart-Jones GB, Tarry MJ, Dejnirattisai W, Siebold C, Koch M, Stuart DI, Harlos K, Malasit P, Screaton G et al. 2007. Humidity control as a strategy for lattice optimization applied to crystals of HLA-A*1101 complexed with variant peptides from dengue virus. Acta Crystallogr Sect F Struct Biol Cryst Commun, 63 (Pt 5), pp. 386-392. | Show Abstract | Read more

T-cell recognition of the antigenic peptides presented by MHC class I molecules normally triggers protective immune responses, but can result in immune enhancement of disease. Cross-reactive T-cell responses may underlie immunopathology in dengue haemorrhagic fever. To analyze these effects at the molecular level, the functional MHC class I molecule HLA-A*1101 was crystallized bound to six naturally occurring peptide variants from the dengue virus NS3 protein. The crystals contained high levels of solvent and required optimization of the cryoprotectant and dehydration protocols for each complex to yield well ordered diffraction, a process that was facilitated by the use of a free-mounting system.

Matadeen R, Hon WC, Heath JK, Jones EY, Fuller S. 2007. The dynamics of signal triggering in a gp130-receptor complex. Structure, 15 (4), pp. 441-448. | Show Abstract | Read more

gp130 is a shared signal-transducing membrane-associated receptor for several hematopoietic cytokines. The 30 A resolution cryo-electron microscopy (cryo-EM) structure of the Interleukin 11(IL-11)-IL-11 Receptor-gp130 extracellular complex reveals the architecture and dynamics of this gp130-containing signaling complex. Normal-mode analysis reveals a repertoire of conformational changes that could function in signal triggering. This suggests a concerted mechanism of signaling involving all the components of the complex. This could provide a general mechanism of signal transfer for cytokines utilizing the JAK-STAT signaling cascade.

Chang VT, Crispin M, Aricescu AR, Harvey DJ, Nettleship JE, Fennelly JA, Yu C, Boles KS, Evans EJ, Stuart DI et al. 2007. Glycoprotein structural genomics: solving the glycosylation problem. Structure, 15 (3), pp. 267-273. | Show Abstract | Read more

Glycoproteins present special problems for structural genomic analysis because they often require glycosylation in order to fold correctly, whereas their chemical and conformational heterogeneity generally inhibits crystallization. We show that the "glycosylation problem" can be solved by expressing glycoproteins transiently in mammalian cells in the presence of the N-glycosylation processing inhibitors, kifunensine or swainsonine. This allows the correct folding of the glycoproteins, but leaves them sensitive to enzymes, such as endoglycosidase H, that reduce the N-glycans to single residues, enhancing crystallization. Since the scalability of transient mammalian expression is now comparable to that of bacterial systems, this approach should relieve one of the major bottlenecks in structural genomic analysis.

Zaccai NR, May AP, Robinson RC, Burtnick LD, Crocker PR, Brossmer R, Kelm S, Jones EY. 2007. Crystallographic and in silico analysis of the sialoside-binding characteristics of the Siglec sialoadhesin. J Mol Biol, 365 (5), pp. 1469-1479. | Show Abstract | Read more

The Siglec family of receptors mediates cell-surface interactions through recognition of sialylated glycoconjugates. Previously reported structures of the N-terminal domain of the Siglec sialoadhesin (SnD1) in complex with various sialic acid analogs revealed the structural template for sialic acid binding. To characterize further the carbohydrate-binding properties, we have determined the crystal structures of SnD1 in the absence of ligand, and in complex with 2-benzyl-Neu5NPro and 2-benzyl-Neu5NAc. These structures reveal that SnD1 undergoes very few structural changes on ligand binding and detail how two novel classes of sialic acid analogs bind, one of which unexpectedly can induce Siglec dimerization. In conjunction with in silico analysis, this set of structures informs us about the design of putative ligands with enhanced binding affinities and specificities to different Siglecs, and provides data with which to test the effectiveness of different computational drug design protocols.

Harkiolaki M, Gilbert RJ, Jones EY, Feller SM. 2006. The C-terminal SH3 domain of CRKL as a dynamic dimerization module transiently exposing a nuclear export signal. Structure, 14 (12), pp. 1741-1753. | Show Abstract | Read more

CRKL plays essential roles in cell signaling. It consists of an N-terminal SH2 domain followed by two SH3 domains. SH2 and SH3N bind to signaling proteins, but the function of the SH3C domain has remained largely enigmatic. We show here that the SH3C of CRKL forms homodimers in protein crystals and in solution. Evidence for dimer formation of full-length CRKL is also presented. In the SH3C dimer, a nuclear export signal (NES) is mostly buried under the domain surface. The same is true for a monomeric SH3C obtained under different crystallization conditions. Interestingly, partial SH3 unfolding, such as occurs upon dimer/monomer transition, produces a fully-accessible NES through translocation of a single beta strand. Our results document the existence of an SH3 domain dimer formed through exchange of the first SH3 domain beta strand and suggest that partial unfolding of the SH3C is important for the relay of information in vivo.

Hourigan CS, Harkiolaki M, Peterson NA, Bell JI, Jones EY, O'Callaghan CA. 2006. The structure of the human allo-ligand HLA-B*3501 in complex with a cytochrome p450 peptide: steric hindrance influences TCR allo-recognition. Eur J Immunol, 36 (12), pp. 3288-3293. | Show Abstract | Read more

Virus-specific T cell populations have been implicated in allo-recognition. The subdominant T cell receptor JL12 recognizes both HLA-B*0801 presenting the Epstein-Barr virus-derived peptide FLRGRAYGL and also HLA-B*3501 presenting the cytochrome p450 self peptide KPIVVLHGY. This cross-reactivity could promote the rejection of HLA-B*3501-positive cells in Epstein-Barr virus-exposed HLA-B*0801 recipients. LC13, the dominant TCR against the HLA-B*0801:FLRGRAYGL complex, fails to recognize HLA-B*3501:KPIVVLHGY. We report the 1.75-Angstrom resolution crystal structure of the human allo-ligand HLA-B*3501:KPIVVLHGY. Similarities between this structure and that of HLA-B*0801:FLRGRAYGL may facilitate cross-recognition by JL12. Moreover, the elevated peptide position in HLA-B*3501:KPIVVLHGY would provide steric hindrance to LC13, preventing it from interacting in the manner in which it interacts with HLA-B*0801:FLRGRAYGL. These findings are relevant to understanding the basis of T cell cross-reactivity in allo-recognition, optimal transplant donor-recipient matching and developing specific molecular inhibitors of allo-recognition.

Aricescu AR, Lu W, Jones EY. 2006. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr D Biol Crystallogr, 62 (Pt 10), pp. 1243-1250. | Show Abstract | Read more

Most proteins for structural biology studies are produced by high-level expression in Escherichia coli. However, prokaryotic based expression systems fail to generate correctly folded functional forms of many proteins and hence a variety of eukaryotic based expression systems have been developed. Of these, yeast and baculovirus-infected insect cells currently represent the expression systems of choice for structural biologists. Here, protocols for a simple, fast and affordable method for transient protein expression in mammalian cells are reported. The results demonstrate that it combines several features necessary for the production of suitable samples for structural biology, in particular protein crystallography, namely high protein yield, straightforward purification, selenomethionine incorporation and control of N-linked glycosylation. The system is suitable for use in conventional laboratories or can be implemented in a medium- or high-throughput pipeline.

Aricescu AR, Assenberg R, Bill RM, Busso D, Chang VT, Davis SJ, Dubrovsky A, Gustafsson L, Hedfalk K, Heinemann U et al. 2006. Eukaryotic expression: developments for structural proteomics. Acta Crystallogr D Biol Crystallogr, 62 (Pt 10), pp. 1114-1124. | Show Abstract | Read more

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.

Banci L, Bertini I, Cusack S, de Jong RN, Heinemann U, Jones EY, Kozielski F, Maskos K, Messerschmidt A, Owens R et al. 2006. First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value. Acta Crystallogr D Biol Crystallogr, 62 (Pt 10), pp. 1208-1217. | Show Abstract | Read more

The EC 'Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, approximately 170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples.

Evans EJ, Castro MA, O'Brien R, Kearney A, Walsh H, Sparks LM, Tucknott MG, Davies EA, Carmo AM, van der Merwe PA et al. 2006. Crystal structure and binding properties of the CD2 and CD244 (2B4)-binding protein, CD48. J Biol Chem, 281 (39), pp. 29309-29320. | Show Abstract | Read more

The structural analysis of surface proteins belonging to the CD2 subset of the immunoglobulin superfamily has yielded important insights into transient cellular interactions. In mice and rats, CD2 and CD244 (2B4), which are expressed predominantly on T cells and natural killer cells, respectively, bind the same, broadly expressed ligand, CD48. Structures of CD2 and CD244 have been solved previously, and we now present the structure of the receptor-binding domain of rat CD48. The receptor-binding surface of CD48 is unusually flat, as in the case of rat CD2, and shares a high degree of electrostatic complementarity with the equivalent surface of CD2. The relatively simple arrangement of charged residues and this flat topology explain why CD48 cross-reacts with CD2 and CD244 and, in rats, with the CD244-related protein, 2B4R. Comparisons of modeled complexes of CD2 and CD48 with the complex of human CD2 and CD58 are suggestive of there being substantial plasticity in the topology of ligand binding by CD2. Thermodynamic analysis of the native CD48-CD2 interaction indicates that binding is driven by equivalent, weak enthalpic and entropic effects, in contrast to the human CD2-CD58 interaction, for which there is a large entropic barrier. Overall, the structural and biophysical comparisons of the CD2 homologues suggest that the evolutionary diversification of interacting cell surface proteins is rapid and constrained only by the requirement that binding remains weak and specific.

Gillespie GM, Stewart-Jones G, Rengasamy J, Beattie T, Bwayo JJ, Plummer FA, Kaul R, McMichael AJ, Easterbrook P, Dong T et al. 2006. Strong TCR conservation and altered T cell cross-reactivity characterize a B*57-restricted immune response in HIV-1 infection. J Immunol, 177 (6), pp. 3893-3902. | Show Abstract

HLA-B*57 is associated with slower disease progression to AIDS, and CD8+ T cell responses to B*57-restricted epitopes are thought to contribute to this protective effect. In this study, we evaluate the B*57-restricted p24 KAFSPEVIPMF (KF11) immune response which is immunodominant during chronic infection. Previously, we observed that the KF11 clade variants KGFNPEVIPMF [A2G,S4N] and KAFNPEIIMPF [S4N,V7I], sharing a position 4 mutation, are differentially recognized by KF11-specific T cells. By combining structural and cellular studies, we now demonstrate that the KF11 and [A2G,S4N] epitopes induce distinct functional responses in [A2G,S4N] and KF11-specific T cells, respectively, despite minimal structural differences between the individual B*57-peptide complexes. Recently, we also elucidated the highly distinct structure of KF11 in complex with B*5703, and have now characterized the CD8+ T cell repertoire recognizing this epitope. We now report striking features of TCR conservation both in terms of TCR Valpha and Vbeta chain usage, and throughout the hypervariable region. Collectively, our findings highlight unusual features of the B*5701/B*5703-KF11-specific immune responses which could influence disease progression and that might be important to consider when designing future vaccine regimens.

Roche P, Brown J, Denley A, Forbes BE, Wallace JC, Jones EY, Esnouf RM. 2006. Computational model for the IGF-II/IGF2r complex that is predictive of mutational and surface plasmon resonance data. Proteins, 64 (3), pp. 758-768. | Show Abstract | Read more

Insulin-like growth factors (IGFs) are key regulators of cell proliferation, differentiation, and transformation, and are thus pivotal in cancer, especially breast, prostate, and colon neoplasm. Their potent mitogenic and anti-apoptotic actions depend primarily on their availability to bind to the signaling IGF cell surface receptors. One mechanism by which IGF-II availability is thought to be modulated is by binding to the nonsignaling IGF-II receptor (IGF2R). This binding is essentially mediated by domain 11 in the multidomain IGF2R extracellular region. The crystal structure of domain 11 of the human IGF-II receptor (IGF2R-d11) has identified a putative IGF-II binding site, and a nuclear magnetic resonance (NMR) solution structure for the IGF-II ligand has also been characterized. These structures have now been used to model in silico the protein-protein interaction between IGF-II and IGF2R-d11 using the program 3D-Dock. Because the IGF-II data comprise an ensemble of 20 structures, all of which satisfy the NMR constraints, the docking procedure was applied to each member of the ensemble. Only those models in which residue Ile1572 of IGF2R-d11, known to be essential for the binding of IGF-II, was at the interface were considered further. These plausible complexes were then critically assessed using an array of analysis techniques including consideration of additional mutagenesis data. One model was strongly supported by these analyses and is discussed here in detail. Furthermore, we demonstrate in vitro experimental support for this model by studying the binding of chimeras of IGF-I and IGF-II to IGF2R fragments.

Crispin M, Harvey DJ, Chang VT, Yu C, Aricescu AR, Jones EY, Davis SJ, Dwek RA, Rudd PM. 2006. Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway. Glycobiology, 16 (8), pp. 748-756. | Show Abstract | Read more

A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.

Zaccheo OJ, Prince SN, Miller DM, Williams C, Kemp CF, Brown J, Jones EY, Catto LE, Crump MP, Hassan AB. 2006. Kinetics of insulin-like growth factor II (IGF-II) interaction with domain 11 of the human IGF-II/mannose 6-phosphate receptor: function of CD and AB loop solvent-exposed residues. J Mol Biol, 359 (2), pp. 403-421. | Show Abstract | Read more

Ligands of the IGF-II/mannose 6-phosphate receptor (IGF2R) include IGF-II and mannose 6-phosphate modified proteins. Disruption of the negative regulatory effects of IGF2R on IGF-II-induced growth can lead to embryonic lethality and cancer promotion. Of the 15 IGF2R extracellular domains, domains 1-3 and 11 are known to have a conserved beta-barrel structure similar to that of avidin and the cation-dependent mannose 6-phosphate receptor, yet only domain 11 binds IGF-II with high specificity and affinity. In order to define the functional basis of this critical biological interaction, we performed alanine mutagenesis of structurally determined solvent-exposed loop residues of the IGF-II-binding site of human domain 11, expressed these mutant forms in Pichia pastoris, and determined binding kinetics with human IGF-II using isothermal calorimetry and surface plasmon resonance with transition state thermodynamics. Two hydrophobic residues in the CD loop (F1567 and I1572) were essential for binding, with a further non-hydrophobic residue (T1570) that slows the dissociation rate. Aside from alanine mutations of AB loop residues that decrease affinity by modifying dissociation rates (e.g. Y1542), a novel mutation (E1544A) of the AB loop enhanced affinity by threefold compared to wild-type. Conversion from an acidic to a basic residue at this site (E1544K) results in a sixfold enhancement of affinity via modification principally of the association rate, with enhanced salt-dependence, decreased entropic barrier and retained specificity. These data suggest that a functional hydrophobic binding site core is formed by I1572 and F1567 located in the CD loop, which initially anchors IGF-II. Within the AB loop, residues normally act to either stabilise or function as negative regulators of the interaction. These findings have implications for the molecular architecture and evolution of the domain 11 IGF-II-binding site, and the potential interactions with other domains of IGF2R.

Glithero A, Tormo J, Doering K, Kojima M, Jones EY, Elliott T. 2006. The crystal structure of H-2D(b) complexed with a partial peptide epitope suggests a major histocompatibility complex class I assembly intermediate. J Biol Chem, 281 (18), pp. 12699-12704. | Show Abstract | Read more

In the absence of bound peptide ligands, major histocompatibility complex (MHC) class I molecules are unstable. In an attempt to determine the minimum requirement for peptide-dependent MHC class I stabilization, we have used short synthetic peptides derived from the Sendai virus nucleoprotein epitope (residues 324-332, 1FAPGNYPAL9) to promote its folding in vitro of H-2D(b). We found that H-2D(b) can be stabilized by the pentapeptide 5NYPAL9, which is equivalent to the C-terminal portion of the optimal nonapeptide and includes both the P5 and P9 anchor residues. We have crystallized the complex of the H-2D(b) molecule with the pentamer and determined the structure to show how a quasi-stable MHC class I molecule can be formed by occupancy of a single binding pocket in the peptide-binding groove.

Jones EY, Fugger L, Strominger JL, Siebold C. 2006. MHC class II proteins and disease: a structural perspective. Nat Rev Immunol, 6 (4), pp. 271-282. | Show Abstract | Read more

MHC class II molecules on the surface of antigen-presenting cells display a range of peptides for recognition by the T-cell receptors of CD4+ T helper cells. Therefore, MHC class II molecules are central to effective adaptive immune responses, but conversely, genetic and epidemiological data have implicated these molecules in the pathogenesis of autoimmune diseases. Indeed, the strength of the associations between particular MHC class II alleles and disease render them the main genetic risk factors for autoimmune disorders such as type 1 diabetes. Here, we discuss the insights that the crystal structures of MHC class II molecules provide into the molecular mechanisms by which sequence polymorphisms might contribute to disease susceptibility.

Gadola SD, Koch M, Marles-Wright J, Lissin NM, Shepherd D, Matulis G, Harlos K, Villiger PM, Stuart DI, Jakobsen BK et al. 2006. Structure and binding kinetics of three different human CD1d-alpha-galactosylceramide-specific T cell receptors. J Exp Med, 203 (3), pp. 699-710. | Show Abstract | Read more

Invariant human TCR Valpha24-Jalpha18+/Vbeta11+ NKT cells (iNKT) are restricted by CD1d-alpha-glycosylceramides. We analyzed crystal structures and binding characteristics for an iNKT TCR plus two CD1d-alpha-GalCer-specific Vbeta11+ TCRs that use different TCR Valpha chains. The results were similar to those previously reported for MHC-peptide-specific TCRs, illustrating the versatility of the TCR platform. Docking TCR and CD1d-alpha-GalCer structures provided plausible insights into their interaction. The model supports a diagonal orientation of TCR on CD1d and suggests that complementarity determining region (CDR)3alpha, CDR3beta, and CDR1beta interact with ligands presented by CD1d, whereas CDR2beta binds to the CD1d alpha1 helix. This docking provides an explanation for the dominant usage of Vbeta11 and Vbeta8.2 chains by human and mouse iNKT cells, respectively, for recognition of CD1d-alpha-GalCer.

Mongkolsapaya J, Duangchinda T, Dejnirattisai W, Vasanawathana S, Avirutnan P, Jairungsri A, Khemnu N, Tangthawornchaikul N, Chotiyarnwong P, Sae-Jang K et al. 2006. T cell responses in dengue hemorrhagic fever: are cross-reactive T cells suboptimal? J Immunol, 176 (6), pp. 3821-3829. | Show Abstract

Dengue virus infection poses a growing public health and economic burden in a number of tropical and subtropical countries. Dengue circulates as a number of quasispecies, which can be divided by serology into four groups or serotypes. An interesting feature of Dengue, recognized over five decades ago, is that most severe cases that show hemorrhagic fever are not suffering from a primary infection. Instead, they are reinfected with a virus of different serotype. This observation poses considerable problems in vaccine design, and it is therefore imperative to gain a full understanding of the mechanisms underlying this immunological enhancement of disease. In this study, we examined a T cell epitope restricted by HLA-A*24, a major MHC class I allele, in Southeast Asia in a cohort of children admitted to a hospital with acute Dengue infection. The cytokine profiles and the degranulation capacity of T cells generated to this epitope are defined and compared across different viral serotypes. Cross-reactive Dengue-specific T cells seem to show suboptimal degranulation but high cytokine production, which may contribute to the development of the vascular leak characteristic of Dengue hemorrhagic fever.

Aricescu AR, Hon WC, Siebold C, Lu W, van der Merwe PA, Jones EY. 2006. Molecular analysis of receptor protein tyrosine phosphatase mu-mediated cell adhesion. EMBO J, 25 (4), pp. 701-712. | Show Abstract | Read more

Type IIB receptor protein tyrosine phosphatases (RPTPs) are bi-functional cell surface molecules. Their ectodomains mediate stable, homophilic, cell-adhesive interactions, whereas the intracellular catalytic regions can modulate the phosphorylation state of cadherin/catenin complexes. We describe a systematic investigation of the cell-adhesive properties of the extracellular region of RPTPmu, a prototypical type IIB RPTP. The crystal structure of a construct comprising its N-terminal MAM (meprin/A5/mu) and Ig domains was determined at 2.7 A resolution; this assigns the MAM fold to the jelly-roll family and reveals extensive interactions between the two domains, which form a rigid structural unit. Structure-based site-directed mutagenesis, serial domain deletions and cell-adhesion assays allowed us to identify the four N-terminal domains (MAM, Ig, fibronectin type III (FNIII)-1 and FNIII-2) as a minimal functional unit. Biophysical characterization revealed at least two independent types of homophilic interaction which, taken together, suggest that there is the potential for formation of a complex and possibly ordered array of receptor molecules at cell contact sites.

Iversen AK, Stewart-Jones G, Learn GH, Christie N, Sylvester-Hviid C, Armitage AE, Kaul R, Beattie T, Lee JK, Li Y et al. 2006. Conflicting selective forces affect T cell receptor contacts in an immunodominant human immunodeficiency virus epitope. Nat Immunol, 7 (2), pp. 179-189. | Show Abstract | Read more

Cytotoxic T lymphocytes (CTLs) are critical for the control of human immunodeficiency virus, but containment of virus replication can be undermined by mutations in CTL epitopes that lead to virus escape. We analyzed the evolution in vivo of an immunodominant, HLA-A2-restricted CTL epitope and found two principal, diametrically opposed evolutionary pathways that exclusively affect T cell-receptor contact residues. One pathway was characterized by acquisition of CTL escape mutations and the other by selection for wild-type amino acids. The pattern of CTL responses to epitope variants shaped which variant(s) prevailed in the virus population. The pathways notably influenced the amount of plasma virus, as patients with efficient CTL selection had lower plasma viral loads than did patients without efficient selection. Thus, viral escape from CTL responses does not necessarily correlate with disease progression.

Stuart DI, Jones EY, Wilson KS, Daenke S. 2006. SPINE: Structural Proteomics IN Europe – the best of both worlds Acta Crystallographica Section D Biological Crystallography, 62 (10), pp. ii-i. | Read more

Esnouf RM, Love CA, Harlos K, Stuart DI, Jones EY. 2006. Structure determination of human semaphorin 4D as an example of the use of MAD in non-optimal cases. Acta Crystallogr D Biol Crystallogr, 62 (Pt 1), pp. 108-115. | Show Abstract | Read more

Semaphorins are an important class of signalling molecules involved in axon guidance, immune function and angiogenesis. They are characterized by having an extracellular sema domain of about 500 residues. The steps involved in the determination of the structure of human semaphorin 4D are described here as a case study of selenium MAD phasing in a difficult case with low symmetry, moderate diffraction and low selenium content. A particular feature of this study was the large number of diffraction images required to give data of sufficient quality for structure determination and these data are re-analyzed here to investigate the effects of radiation damage on eventual data quality and to suggest strategies for successful MAD phasing in similar difficult cases.

Petry CJ, Ong KK, Wingate DL, Brown J, Scott CD, Jones EY, Pembrey ME, Dunger DB, Alspac Study Team. 2005. Genetic variation in the type 2 insulin-like growth factor receptor gene and disparity in childhood height. Growth Horm IGF Res, 15 (6), pp. 363-368. | Show Abstract | Read more

OBJECTIVE: The type 2 insulin-like growth factor receptor (IGF2R) is thought to regulate insulin-like growth factor-II (IGF-II) bioavailability by degrading it in the lysosomes after uptake. We hypothesised that polymorphisms in the IGF2R gene could alter size at birth and childhood growth. DESIGN AND METHODS: The hypothesis was tested in a normal birth cohort (Avon Longitudinal Study of Parents and Children) by genotyping the IGF2R gene gly1619arg polymorphism, which causes a non-conservative amino acid change in the IGF-II binding region, using PCR and restriction fragment length polymorphism analysis. RESULTS: The IGF2R gly1619arg genotype was not associated with any measure of size at birth, but A/A homozygotes grew more slowly, as determined by their change in height standard deviation scores (SDS) over the first three years (-0.70 (0.72); n = 12), than G/G homozygotes (0.00 (1.09); n = 561) (p = 0.03). They remained shorter during childhood and by the age of 7 years respective height SDS were: 0.73 (1.02) (n = 12) and 0.01 (0.99) (n = 634) (p = 0.01). These height differences persisted after adjusting for parental heights and gender. There were no detectable differences in weights at 7 years. CONCLUSION: Allelic variation in the gly1619arg SNP of the IGF2R gene is associated with disparity in childhood stature which could reflect altered binding of IGF-II to its receptor.

Siebold C, Berrow N, Walter TS, Harlos K, Owens RJ, Stuart DI, Terman JR, Kolodkin AL, Pasterkamp RJ, Jones EY. 2005. High-resolution structure of the catalytic region of MICAL (molecule interacting with CasL), a multidomain flavoenzyme-signaling molecule. Proc Natl Acad Sci U S A, 102 (46), pp. 16836-16841. | Show Abstract | Read more

Semaphorins are extracellular cell guidance cues that govern cytoskeletal dynamics during neuronal and vascular development. MICAL (molecule interacting with CasL) is a multidomain cytosolic protein with a putative flavoprotein monooxygenase (MO) region required for semaphorin-plexin repulsive axon guidance. Here, we report the 1.45-A resolution crystal structure of the FAD-containing MO domain of mouse MICAL-1 (residues 1-489). The topology most closely resembles that of the NADPH-dependent flavoenzyme p-hydroxybenzoate hydroxylase (PHBH). Comparison of structures before and after reaction with NADPH reveals that, as in PHBH, the flavin ring can switch between two discrete positions. In contrast with other MOs, this conformational switch is coupled with the opening of a channel to the active site, suggestive of a protein substrate. In support of this hypothesis, distinctive structural features highlight putative protein-binding sites in suitable proximity to the active site entrance. The unusual juxtaposition of this N-terminal MO (hydroxylase) activity with the characteristics of a multiprotein-binding scaffold exhibited by the C-terminal portion of the MICALs represents a unique combination of functionality to mediate signaling.

Friese MA, Jones EY, Fugger L. 2005. MHC II molecules in inflammatory diseases: interplay of qualities and quantities. Trends Immunol, 26 (11), pp. 559-561. | Show Abstract | Read more

It is generally accepted that MHC II molecules confer susceptibility to inflammatory diseases because of the different abilities they possess for binding and presenting peptides to T cells. A new study suggests that the level of MHC II gene expression is also a risk factor for such diseases. It shows that a polymorphism in the promoter of the MHC II transactivator (MHC2TA) gene (which encodes CIITA), leads to reduced MHC2TA expression, and hence reduced production of MHC II molecules. This predisposes to rheumatoid arthritis, multiple sclerosis and myocardial infarction.

Lamagna C, Meda P, Mandicourt G, Brown J, Gilbert RJ, Jones EY, Kiefer F, Ruga P, Imhof BA, Aurrand-Lions M. 2005. Dual interaction of JAM-C with JAM-B and alpha(M)beta2 integrin: function in junctional complexes and leukocyte adhesion. Mol Biol Cell, 16 (10), pp. 4992-5003. | Show Abstract | Read more

The junctional adhesion molecules (JAMs) have been recently described as interendothelial junctional molecules and as integrin ligands. Here we show that JAM-B and JAM-C undergo heterophilic interaction in cell-cell contacts and that JAM-C is recruited and stabilized in junctional complexes by JAM-B. In addition, soluble JAM-B dissociates soluble JAM-C homodimers to form JAM-B/JAM-C heterodimers. This suggests that the affinity of JAM-C monomers to form dimers is higher for JAM-B than for JAM-C. Using antibodies against JAM-C, the formation of JAM-B/JAM-C heterodimers can be abolished. This liberates JAM-C from its vascular binding partner JAM-B and makes it available on the apical side of vessels for interaction with its leukocyte counter-receptor alpha(M)beta2 integrin. We demonstrate that the modulation of JAM-C localization in junctional complexes is a new regulatory mechanism for alpha(M)beta2-dependent adhesion of leukocytes.

Sajnani G, Aricescu AR, Jones EY, Gallagher J, Alete D, Stoker A. 2005. PTPsigma promotes retinal neurite outgrowth non-cell-autonomously. J Neurobiol, 65 (1), pp. 59-71. | Show Abstract | Read more

The receptor-like protein tyrosine phosphatase (RPTP) PTPsigma controls the growth and targeting of retinal axons, both in culture and in ovo. Although the principal actions of PTPsigma have been thought to be cell-autonomous, the possibility that RPTPs related to PTPsigma also have non-cell-autonomous signaling functions during axon development has also been supported genetically. Here we report that a cell culture substrate made from purified PTPsigma ectodomains supports retinal neurite outgrowth in cell culture. We show that a receptor for PTPsigma must exist on retinal axons and that binding of PTPsigma to this receptor does not require the known, heparin binding properties of PTPsigma. The neurite-promoting potential of PTPsigma ectodomains requires a basic amino acid domain, previously demonstrated in vitro as being necessary for ligand binding by PTPsigma. Furthermore, we demonstrate that heparin and oligosaccharide derivatives as short as 8mers, can specifically block neurite outgrowth on the PTPsigma substrate, by competing for binding to this same domain. This is the first direct evidence of a non-cell-autonomous, neurite-promoting function of PTPsigma and of a potential role for heparin-related oligosaccharides in modulating neurite promotion by an RPTP.

Stewart-Jones GB, Gillespie G, Overton IM, Kaul R, Roche P, McMichael AJ, Rowland-Jones S, Jones EY. 2005. Structures of three HIV-1 HLA-B*5703-peptide complexes and identification of related HLAs potentially associated with long-term nonprogression. J Immunol, 175 (4), pp. 2459-2468. | Show Abstract

Long-term nonprogression during acute HIV infection has been strongly associated with HLA-B*5701 or HLA-B*5703. In this study, we present the high resolution crystal structures of HLA-B*5703 complexes with three HIV-1 epitopes: ISPRTLNAW (ISP), KAFSPEVIPMF (KAF-11), and KAFSPEVI (KAF-8). These reveal peptide anchoring at position 2 and their C termini. The different peptide lengths and primary sequences are accommodated by variation in the specific contacts made to the HLA-B*5703, flexibility in water structure, and conformational adjustment of side chains within the peptide-binding groove. The peptides adopt markedly different conformations, and trap variable numbers of water molecules, near a cluster of tyrosine side chains located in the central region of the peptide-binding groove. The KAF-11 epitope completely encompasses the shorter KAF-8 epitope but the peptides are presented in different conformations; the KAF-11 peptide arches out of the peptide-binding groove, exposing a significant main chain surface area. Bioinformatic analysis of the MHC side chains observed to contribute to the peptide anchor specificity, and other specific peptide contacts, reveals HLA alleles associated with long-term nonprogression and a number of related HLA alleles that may share overlapping peptide repertoires with HLA-B*5703 and thus may display a similar capacity for efficient immune control of HIV-1 infection.

Koch M, Stronge VS, Shepherd D, Gadola SD, Mathew B, Ritter G, Fersht AR, Besra GS, Schmidt RR, Jones EY, Cerundolo V. 2005. The crystal structure of human CD1d with and without alpha-galactosylceramide. Nat Immunol, 6 (8), pp. 819-826. | Show Abstract | Read more

The glycolipid alpha-galactosylceramide binds with high affinity to CD1d and stimulates natural killer T cells. Here we report the crystal structure of human CD1d in complex with synthetic alpha-galactosylceramide at a resolution of 3.0 A. The structure shows a tightly fit lipid in the CD1d binding groove, with the sphingosine chain bound in the C' pocket and the longer acyl chain anchored in the A' pocket. We also present the CD1d structure without lipid, which has a more open conformation of the binding groove, suggesting a dual conformation of CD1d in which the 'open' conformation is more able to load lipids. These structures provide clues as to how CD1 molecules load glycolipids as well as data to guide the design of new therapeutic agents.

Walter TS, Diprose JM, Mayo CJ, Siebold C, Pickford MG, Carter L, Sutton GC, Berrow NS, Brown J, Berry IM et al. 2005. A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallogr D Biol Crystallogr, 61 (Pt 6), pp. 651-657. | Show Abstract | Read more

Crystallization trials at the Division of Structural Biology in Oxford are now almost exclusively carried out using a high-throughput workflow implemented in the Oxford Protein Production Facility. Initial crystallization screening is based on nanolitre-scale sitting-drop vapour-diffusion experiments (typically 100 nl of protein plus 100 nl of reservoir solution per droplet) which use standard crystallization screening kits and 96-well crystallization plates. For 294 K crystallization trials the barcoded crystallization plates are entered into an automated storage system with a fully integrated imaging system. These plates are imaged in accordance with a pre-programmed schedule and the resulting digital data for each droplet are harvested into a laboratory information-management system (LIMS), scored by crystal recognition software and displayed for user analysis via a web-based interface. Currently, storage for trials at 277 K is not automated and for imaging the crystallization plates are fed by hand into an imaging system from which the data enter the LIMS. The workflow includes two procedures for nanolitre-scale optimization of crystallization conditions: (i) a protocol for variation of pH, reservoir dilution and protein:reservoir ratio and (ii) an additive screen. Experience based on 592 crystallization projects is reported.

Chen JL, Stewart-Jones G, Bossi G, Lissin NM, Wooldridge L, Choi EM, Held G, Dunbar PR, Esnouf RM, Sami M et al. 2005. Structural and kinetic basis for heightened immunogenicity of T cell vaccines. J Exp Med, 201 (8), pp. 1243-1255. | Show Abstract | Read more

Analogue peptides with enhanced binding affinity to major histocompatibility class (MHC) I molecules are currently being used in cancer patients to elicit stronger T cell responses. However, it remains unclear as to how alterations of anchor residues may affect T cell receptor (TCR) recognition. We correlate functional, thermodynamic, and structural parameters of TCR-peptide-MHC binding and demonstrate the effect of anchor residue modifications of the human histocompatibility leukocyte antigens (HLA)-A2 tumor epitope NY-ESO-1(157-165)-SLLMWITQC on TCR recognition. The crystal structure of the wild-type peptide complexed with a specific TCR shows that TCR binding centers on two prominent, sequential, peptide sidechains, methionine-tryptophan. Cysteine-to-valine substitution at peptide position 9, while optimizing peptide binding to the MHC, repositions the peptide main chain and generates subtly enhanced interactions between the analogue peptide and the TCR. Binding analyses confirm tighter binding of the analogue peptide to HLA-A2 and improved soluble TCR binding. Recognition of analogue peptide stimulates faster polarization of lytic granules to the immunological synapse, reduces dependence on CD8 binding, and induces greater numbers of cross-reactive cytotoxic T lymphocyte to SLLMWITQC. These results provide important insights into heightened immunogenicity of analogue peptides and highlight the importance of incorporating structural data into the process of rational optimization of superagonist peptides for clinical trials.

Jones EY. 2005. Favorite flavors of surfaces. Nat Immunol, 6 (4), pp. 365-366. | Show Abstract | Read more

Different peptide antigens induce T cell repertoires of very different diversity. An analysis of the effect on T cell receptor usage of re-engineering peptide features indicates that a lack of prominent side chains presented for recognition limits the T cell repertoire.

Holmes S, Friese MA, Siebold C, Jones EY, Bell J, Fugger L. 2005. Multiple sclerosis: MHC associations and therapeutic implications. Expert Rev Mol Med, 7 (3), pp. 1-17. | Show Abstract | Read more

Multiple sclerosis (MS) is an autoimmune disease with an important genetic component. The strongest genetic association is with the major histocompatibility complex (MHC) region. Several MHC alleles predispose to the disease, the most prominent of which are certain alleles in the HLA-DR2 haplotype. Functional and structural studies have helped to explain the molecular basis of these associations. Although there is currently no curative treatment for MS, an increased understanding of the disease has aided the design of immunotherapies that act on the immune system more specifically than the longstanding drugs. Many of these therapies work at the antigen-specific level, disrupting the interaction between T-cell receptors and MHC molecules that leads to disease.

Mayo CJ, Diprose JM, Walter TS, Berry IM, Wilson J, Owens RJ, Jones EY, Harlos K, Stuart DI, Esnouf RM. 2005. Benefits of automated crystallization plate tracking, imaging, and analysis. Structure, 13 (2), pp. 175-182. | Show Abstract | Read more

We describe the design of a database and software for managing and organizing protein crystallization data. We also outline the considerations behind the design of a fast web interface linking protein production data, crystallization images, and automated image analysis. The database and associated interfaces underpin the Oxford Protein Production Facility (OPPF) crystallization laboratory, collecting, in a routine and automatic manner, up to 100,000 images per day. Over 17 million separate images are currently held in this database. We discuss the substantial scientific benefits automated tracking, imaging, and analysis of crystallizations offers to the structural biologist: analysis of the time course of the trial and easy analysis of trials with related crystallization conditions. Features of this system address requirements common to many crystallographic laboratories that are currently setting up (semi-)automated crystallization imaging systems.

Stewart-Jones GB, di Gleria K, Kollnberger S, McMichael AJ, Jones EY, Bowness P. 2005. Crystal structures and KIR3DL1 recognition of three immunodominant viral peptides complexed to HLA-B*2705. Eur J Immunol, 35 (2), pp. 341-351. | Show Abstract | Read more

We have solved the crystal structures of three HLA-B*2705-peptide complexes with the immunodominant viral peptides: EBV EBNA3C 258-266 (RRIYDLIEL), influenza (flu) nucleoprotein NP383-391 (SRYWAIRTR), and HIV gag 264-273 (KRWIILGLNK). Long-term non-progression during HIV infection has been associated with presentation by HLA-B*2705, and T cell recognition, of the highly immunodominant KRWIILGLNK peptide. The tight hydrogen-bonding network observed between the HLA-B*2705 B-pocket and the peptide P2 arginine guanadinium anchor explains why mutation of this residue during HIV infection results in loss of peptide binding, immune escape and progression to AIDS. Prominent, solvent-exposed structures within these peptides may participate in generating T cell responses to these immunodominant epitopes. In the HLA-B*2705 complex with flu NP383-391, the amino acid side chains of residues 4, 7 and 8 are solvent-exposed whilst in the HIV decamer, the main-chain bulges into the solvent around P7. Thus, HLA-B*2705 presents viral peptides in a range of conformations. Tetrameric complexes of HLA-B*2705 with the HIV and flu but not EBV peptides bound strongly to the killer-Ig-like receptor (KIR)3DL1. Substitution of EBV P8 glutamate to threonine allowed recognition by KIR3DL1. In the HLA-B*2705-EBV structure the P8 glutamate side chain is solvent-exposed and may inhibit KIR3DL1 binding through electrostatic forces.

Dong T, Stewart-Jones G, Chen N, Easterbrook P, Xu X, Papagno L, Appay V, Weekes M, Conlon C, Spina C et al. 2004. HIV-specific cytotoxic T cells from long-term survivors select a unique T cell receptor. J Exp Med, 200 (12), pp. 1547-1557. | Show Abstract | Read more

HIV-specific cytotoxic T lymphocytes (CTL) are important in controlling HIV replication, but the magnitude of the CTL response does not predict clinical outcome. In four donors with delayed disease progression we identified Vbeta13.2 T cell receptors (TCRs) with very similar and unusually long beta-chain complementarity determining region 3 (CDR3) regions in CTL specific for the immunodominant human histocompatibility leukocyte antigens (HLA)-B8-restricted human immunodeficiency virus-1 (HIV-1) nef epitope, FLKEKGGL (FL8). CTL expressing Vbeta13.2 TCRs tolerate naturally arising viral variants in the FL8 epitope that escape recognition by other CTL. In addition, they expand efficiently in vitro and are resistant to apoptosis, in contrast to FL8-specific CTL using other TCRs. Selection of Vbeta13.2 TCRs by some patients early in the FL8-specific CTL response may be linked with better clinical outcome.

Lee JK, Stewart-Jones G, Dong T, Harlos K, Di Gleria K, Dorrell L, Douek DC, van der Merwe PA, Jones EY, McMichael AJ. 2004. T cell cross-reactivity and conformational changes during TCR engagement. J Exp Med, 200 (11), pp. 1455-1466. | Show Abstract | Read more

All thymically selected T cells are inherently cross-reactive, yet many data indicate a fine specificity in antigen recognition, which enables virus escape from immune control by mutation in infections such as the human immunodeficiency virus (HIV). To address this paradox, we analyzed the fine specificity of T cells recognizing a human histocompatibility leukocyte antigen (HLA)-A2-restricted, strongly immunodominant, HIV gag epitope (SLFNTVATL). The majority of 171 variant peptides tested bound HLA-A2, but only one third were recognized. Surprisingly, one recognized variant (SLYNTVATL) showed marked differences in structure when bound to HLA-A2. T cell receptor (TCR) recognition of variants of these two peptides implied that they adopted the same conformation in the TCR-peptide-major histocompatibility complex (MHC) complex. However, the on-rate kinetics of TCR binding were identical, implying that conformational changes at the TCR-peptide-MHC binding interface occur after an initial permissive antigen contact. These findings have implications for the rational design of vaccines targeting viruses with unstable genomes.

Gherardi E, Love CA, Esnouf RM, Jones EY. 2004. The sema domain. Curr Opin Struct Biol, 14 (6), pp. 669-678. | Show Abstract | Read more

The sema domain was first defined from sequence by Kolodkin and colleagues in the early 1990s, and constitutes the distinctive structural and functional element of semaphorins, their plexin receptors and the receptor tyrosine kinases MET and RON, three protein families with major roles in development, tissue regeneration and cancer. Recently determined crystal structures of two semaphorins (SEMA3A and SEMA4D) and the MET receptor have shown that the sema domain consists of a highly conserved variant form of the seven-blade beta-propeller fold. The structures, however, also suggest differences between these families with respect to the mode of dimerisation and the regions of the domain involved in ligand-receptor interactions. This reflects the considerable plasticity and adaptation of the sema domain in order to meet different binding requirements, properties that may underlie the vast array of ligand-receptor specificities and functions of the semaphorin superfamily.

Lewitzky M, Harkiolaki M, Domart MC, Jones EY, Feller SM. 2004. Mona/Gads SH3C binding to hematopoietic progenitor kinase 1 (HPK1) combines an atypical SH3 binding motif, R/KXXK, with a classical PXXP motif embedded in a polyproline type II (PPII) helix. J Biol Chem, 279 (27), pp. 28724-28732. | Show Abstract | Read more

Hematopoietic progenitor kinase 1 (HPK1) is implicated in signaling downstream of the T cell receptor. Its non-catalytic, C-terminal half contains several prolinerich motifs, which have been shown to interact with different SH3 domain-containing adaptor proteins in vitro. One of these, Mona/Gads, was also shown to bind HPK1 in mouse T cells in vivo. The region of HPK1 that binds to the Mona/Gads C-terminal SH3 domain has been mapped and shows only very limited similarity to a recently identified high affinity binding motif in SLP-76, another T-cell adaptor. Using isothermal titration calorimetry and x-ray crystallography, the binding of the HPK1 motif to Mona/Gads SH3C has now been characterized in molecular detail. The results indicate that although charge interactions through an RXXK motif are essential for complex formation, a PXXP motif in HPK1 strongly complements binding. This unexpected binding mode therefore differs considerably from the previously described interaction of Mona/Gads SH3C with SLP-76. The crystal structure of the complex highlights the great versatility of SH3 domains, which allows interactions with very different proteins. This currently limits our ability to categorize SH3 binding properties by simple rules.

Siebold C, Hansen BE, Wyer JR, Harlos K, Esnouf RE, Svejgaard A, Bell JI, Strominger JL, Jones EY, Fugger L. 2004. Crystal structure of HLA-DQ0602 that protects against type 1 diabetes and confers strong susceptibility to narcolepsy. Proc Natl Acad Sci U S A, 101 (7), pp. 1999-2004. | Show Abstract | Read more

The MHC class II molecule DQ0602 confers strong susceptibility to narcolepsy but dominant protection against type 1 diabetes. The crystal structure of DQ0602 reveals the molecular features underlying these contrasting genetic properties. Structural comparisons to homologous DQ molecules with differential disease associations highlight a previously unrecognized interplay between the volume of the P6 pocket and the specificity of the P9 pocket, which implies that presentation of an expanded peptide repertoire is critical for dominant protection against type 1 diabetes. In narcolepsy, the volume of the P4 pocket appears central to the susceptibility, suggesting that the presentation of a specific peptide population plays a major role.

Batuwangala T, Shepherd D, Gadola SD, Gibson KJ, Zaccai NR, Fersht AR, Besra GS, Cerundolo V, Jones EY. 2004. The crystal structure of human CD1b with a bound bacterial glycolipid. J Immunol, 172 (4), pp. 2382-2388. | Show Abstract

The human MHC class I-like molecule CD1b is distinctive among CD1 alleles in that it is capable of presenting a set of glycolipid species that show a very broad range of variation in the lengths of their acyl chains. A structure of CD1b complexed with relatively short acyl chain glycolipids plus detergent suggested how an interlinked network of channels within the Ag-binding groove could accommodate acyl chain lengths of up to 80 carbons. The structure of CD1b complexed with glucose monomycolate, reported in this study, confirms this hypothesis and illustrates how the distinctive substituents of intracellular bacterial glycolipids can be accommodated. The Ag-binding groove of CD1b is, uniquely among CD1 alleles, partitioned into channels suitable for the compact accommodation of lengthy acyl chains. The current crystal structure illustrates for the first time the binding of a natural bacterial lipid Ag to CD1b and shows how its novel structural features fit this molecule for its role in the immune response to intracellular bacteria.

Barberis D, Artigiani S, Casazza A, Corso S, Giordano S, Love CA, Jones EY, Comoglio PM, Tamagnone L. 2004. Plexin signaling hampers integrin-based adhesion, leading to Rho-kinase independent cell rounding, and inhibiting lamellipodia extension and cell motility. FASEB J, 18 (3), pp. 592-594. | Show Abstract | Read more

Plexins encode receptors for semaphorins, molecular signals guiding cell migration, and axon pathfinding. The mechanisms mediating plexin function are poorly understood. Plexin activation in adhering cells rapidly leads to retraction of cellular processes and cell rounding "cell collapse"). Here we show that, unexpectedly, this response does not require the activity of Rho-dependent kinase (ROCK) nor the contraction of F-actin cables. Interestingly, integrin-based focal adhesive structures are disassembled within minutes upon plexin activation; this is followed by actin depolymerization and, eventually, by cellular collapse. We also show that plexin activation hinders cell attachment to adhesive substrates, blocks the extension of lamellipodia, and thereby inhibits cell migration. We conclude that plexin signaling uncouples cell substrate-adhesion from cytoskeletal dynamics required for cell migration and axon extension.

Batuwangala T, Leduc M, Gibbins JM, Bon C, Jones EY. 2004. Structure of the snake-venom toxin convulxin. Acta Crystallogr D Biol Crystallogr, 60 (Pt 1), pp. 46-53. | Show Abstract | Read more

Snake venoms contain a number of proteins that interact with components of the haemostatic system that promote or inhibit events leading to blood-clot formation. The snake-venom protein convulxin (Cvx) binds glycoprotein (GP) VI, the platelet receptor for collagen, and triggers signal transduction. Here, the 2.7 A resolution crystal structure of Cvx is presented. In common with other members of this snake-venom protein family, Cvx is an alphabeta-heterodimer and conforms to the C-type lectin-fold topology. Comparison with other family members allows a set of Cvx residues that form a concave surface to be putatively implicated in GPVI binding. Unlike other family members, with the exception of flavocetin-A (FL-A), Cvx forms an (alphabeta)(4) tetramer. This oligomeric structure is consistent with Cvx clustering GPVI molecules on the surface of platelets and as a result promoting signal transduction activity. The Cvx structure and the location of the putative binding sites suggest a model for this multimeric signalling assembly.

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Allen M, Heinzmann A, Noguchi E, Abecasis G, Broxholme J, Ponting CP, Bhattacharyya S, Tinsley J, Zhang YM, Holt R et al. 2003. Positional cloning of a novel gene influencing asthma from Chromosome 2q14 NATURE GENETICS, 35 (3), pp. 258-263. | Read more

Moreau-Fauvarque C, Kumanogoh A, Camand E, Jaillard C, Barbin G, Boquet I, Love C, Jones EY, Kikutani H, Lubetzki C et al. 2003. The transmembrane semaphorin Sema4D/CD100, an inhibitor of axonal growth, is expressed on oligodendrocytes and upregulated after CNS lesion. J Neurosci, 23 (27), pp. 9229-9239. | Show Abstract

Semaphorins are a family of secreted and membrane-bound proteins, known to regulate axonal pathfinding. Sema4D, also called CD100, was first isolated in the immune system where it is involved in B and T cell activation. We found that in the mouse, Sema4D is expressed in cells throughout the CNS white matter, with a peak during the myelination period. Double-labeling experiments with different markers of oligodendrocyte lineage such as olig1, olig2, platelet-derived growth factor receptor alpha, and proteolipid protein showed that Sema4D was expressed selectively by oligodendrocytes and myelin. The presence of Sema4D in myelin was confirmed using Western blot. Sema4D expression in myelinating oligodendrocytes was further observed using neuron-oligodendrocyte cocultures. Moreover, using stripe assay, we found that Sema4D is strongly inhibitory for postnatal sensory and cerebellar granule cell axons. This prompted us to examine whether Sema4D expression is modified after CNS injury. At 8 d after spinal cord lesions, Sema4D expression was strongly upregulated in oligodendrocytes at the periphery of the lesion. Sema4D-positive cells were not colabeled with the astrocyte marker GFAP, with the microglial and macrophagic marker isolectin B4, or with NG2, a marker of oligodendrocyte precursors. This upregulation was transient because from 1 month after the lesion, Sema4D expression was back to its normal level. These results indicate that Sema4D is a novel inhibitory factor for axonal regeneration expressed in myelin.

Love CA, Harlos K, Mavaddat N, Davis SJ, Stuart DI, Jones EY, Esnouf RM. 2003. The ligand-binding face of the semaphorins revealed by the high-resolution crystal structure of SEMA4D. Nat Struct Biol, 10 (10), pp. 843-848. | Show Abstract | Read more

Semaphorins, proteins characterized by an extracellular sema domain, regulate axon guidance, immune function and angiogenesis. The crystal structure of SEMA4D (residues 1-657) shows the sema topology to be a seven-bladed beta-propeller, revealing an unexpected homology with integrins. The sema beta-propeller contains a distinctive 77-residue insertion between beta-strands C and D of blade 5. Blade 7 is followed by a domain common to plexins, semaphorins and integrins (PSI domain), which forms a compact cysteine knot abutting the side of the propeller, and an Ig-like domain. The top face of the beta-propeller presents prominent loops characteristic of semaphorins. In addition to limited contact between the Ig-like domains, the homodimer is stabilized through extensive interactions between the top faces in a sector of the beta-propeller used for heterodimerization in integrins. This face of the propeller also mediates ligand binding in integrins, and functional data for semaphorin-receptor interactions map to the equivalent surface.

Shiroishi M, Tsumoto K, Amano K, Shirakihara Y, Colonna M, Braud VM, Allan DS, Makadzange A, Rowland-Jones S, Willcox B et al. 2003. Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G. Proc Natl Acad Sci U S A, 100 (15), pp. 8856-8861. | Show Abstract | Read more

Ig-like transcript 4 (ILT4) (also known as leukocyte Ig-like receptor 2, CD85d, and LILRB2) is a cell surface receptor expressed mainly on myelomonocytic cells, whereas ILT2 (also known as leukocyte Ig-like receptor 1, CD85j, and LILRB1) is expressed on a wider range of immune cells including subsets of natural killer and T cells. Both ILTs contain immunoreceptor tyrosine-based inhibitory receptor motifs in their cytoplasmic tails that inhibit cellular responses by recruiting phosphatases such as SHP-1 (Src homology 2 domain containing tyrosine phosphatase 1). Although these ILTs have been shown to recognize a broad range of classical and nonclassical human MHC class I molecules (MHCIs), their precise binding properties remain controversial. We have used surface plasmon resonance to analyze the interaction of soluble forms of ILT4 and ILT2 with several MHCIs. Although the range of affinities measured was quite broad (Kd = 2-45 microM), some interesting differences were observed. ILT2 generally bound with a 2- to 3-fold higher affinity than ILT4 to the same MHCI. Furthermore, ILT2 and ILT4 bound to HLA-G with a 3- to 4-fold higher affinity than to classical MHCIs, suggesting that ILT/HLA-G recognition may play a dominant role in the regulation of natural killer, T, and myelomonocytic cell activation. Finally, we show that ILT2 and ILT4 effectively compete with CD8 for MHCI binding, raising the possibility that ILT2 modulates CD8+ T cell activation by blocking the CD8 binding as well as by recruiting inhibitory molecules through its immunoreceptor tyrosine-based inhibitory receptor motif.

Stewart-Jones GB, McMichael AJ, Bell JI, Stuart DI, Jones EY. 2003. A structural basis for immunodominant human T cell receptor recognition. Nat Immunol, 4 (7), pp. 657-663. | Show Abstract | Read more

The anti-influenza CD8+ T cell response in HLA-A2-positive adults is almost exclusively directed at residues 58-66 of the virus matrix protein (MP(58-66)). V(beta)17V(alpha)10.2 T cell receptors (TCRs) containing a conserved arginine-serine-serine sequence in complementarity determining region 3 (CDR3) of the V(beta) segment dominate this response. To investigate the molecular basis of immunodominant selection in an outbred population, we have determined the crystal structure of V(beta)17V(alpha)10.2 in complex with MP(58-66)-HLA-A2 at a resolution of 1.4 A. We show that, whereas the TCR typically fits over an exposed side chain of the peptide, in this structure MP(58-66) exposes only main chain atoms. This distinctive orientation of V(beta)17V(alpha)10.2, which is almost orthogonal to the peptide-binding groove of HLA-A2, facilitates insertion of the conserved arginine in V(beta) CDR3 into a notch in the surface of MP(58-66)-HLA-A2. This previously unknown binding mode underlies the immunodominant T cell response.

Harkiolaki M, Lewitzky M, Gilbert RJ, Jones EY, Bourette RP, Mouchiroud G, Sondermann H, Moarefi I, Feller SM. 2003. Structural basis for SH3 domain-mediated high-affinity binding between Mona/Gads and SLP-76. EMBO J, 22 (11), pp. 2571-2582. | Show Abstract | Read more

SH3 domains are protein recognition modules within many adaptors and enzymes. With more than 500 SH3 domains in the human genome, binding selectivity is a key issue in understanding the molecular basis of SH3 domain interactions. The Grb2-like adaptor protein Mona/Gads associates stably with the T-cell receptor signal transducer SLP-76. The crystal structure of a complex between the C-terminal SH3 domain (SH3C) of Mona/Gads and a SLP-76 peptide has now been solved to 1.7 A. The peptide lacks the canonical SH3 domain binding motif P-x-x-P and does not form a frequently observed poly-proline type II helix. Instead, it adopts a clamp-like shape around the circumfence of the SH3C beta-barrel. The central R-x-x-K motif of the peptide forms a 3(10) helix and inserts into a negatively charged double pocket on the SH3C while several other residues complement binding through hydrophobic interactions, creating a short linear SH3C binding epitope of uniquely high affinity. Interestingly, the SH3C displays ion-dependent dimerization in the crystal and in solution, suggesting a novel mechanism for the regulation of SH3 domain functions.

Zaccai NR, Maenaka K, Maenaka T, Crocker PR, Brossmer R, Kelm S, Jones EY. 2003. Structure-guided design of sialic acid-based Siglec inhibitors and crystallographic analysis in complex with sialoadhesin. Structure, 11 (5), pp. 557-567. | Show Abstract | Read more

The Siglec family of receptors mediates cell surface interactions through recognition of sialylated glycoconjugates. The crystal structure of the N-terminal immunoglobulin-like domain of the Siglec sialoadhesin (SnD1) in complex with 2,3-sialyllactose has informed the design of sialic acid analogs (sialosides) that bind Siglecs with significantly enhanced affinities and specificities. Binding assays against sialoadhesin (Sn; Siglec-1), CD22 (Siglec-2), and MAG (Siglec-4) show a 10- to 300-fold reduction in IC(50) values (relative to methyl-alpha-Neu5Ac) for three sialosides bearing aromatic group modifications of the glycerol side chain: Me-alpha-9-N-benzoyl-amino-9-deoxy-Neu5Ac (BENZ), Me-alpha-9-N-(naphthyl-2-carbonyl)-amino-9-deoxy-Neu5Ac (NAP), and Me-alpha-9-N-(biphenyl-4-carbonyl)-amino-9-deoxy-Neu5Ac (BIP). Crystal structures of these sialosides in complex with SnD1 suggest explanations for the differences in specificity and affinity, providing further ideas for compound design of physiological and potentially therapeutic relevance.

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Brown J, Walter TS, Carter L, Abrescia NGA, Aricescu AR, Batuwangala TD, Bird LE, Brown N, Chamberlain PP, Davis SJ et al. 2003. A procedure for setting up high-throughput nanolitre crystallization experiments. II. Crystallization results JOURNAL OF APPLIED CRYSTALLOGRAPHY, 36 (2), pp. 315-318. | Show Abstract | Read more

An initial tranche of results from day-to-day use of a robotic system for setting up 100 nl-scale vapour-diffusion sitting-drop protein crystallizations has been surveyed. The database of over 50 unrelated samples represents a snapshot of projects currently at the stage of crystallization trials in Oxford research groups and as such encompasses a broad range of proteins. The results indicate that the nanolitre-scale methodology consistently identifies more crystallization conditions than traditional hand-pipetting-style methods; however, in a number of cases successful scale-up is then problematic. Crystals grown in the initial 100 nl-scale drops have in the majority of cases allowed useful characterization of x-ray diffraction, either in-house or at synchrotron beamlines. For a significant number of projects, full x-ray diffraction data sets have been collected to 3 Å resolution or better (either in-house or at the synchrotron) from crystals grown at the 100 nl scale. To date, five structures have been determined by molecular replacement directly from such data and a further three from scale-up of conditions established at the nanolitre scale.

O'Callaghan CA, Jones EY. 2003. Structural and energetic aspects of multispecific immune recognition by NKG2D. Structure, 11 (4), pp. 360-361. | Show Abstract | Read more

The multispecific immune receptor NKG2D binds different ligands using a different set of energetically dominant interface residues for each ligand.

Lang HL, Jacobsen H, Ikemizu S, Andersson C, Harlos K, Madsen L, Hjorth P, Sondergaard L, Svejgaard A, Wucherpfennig K et al. 2002. A functional and structural basis for TCR cross-reactivity in multiple sclerosis. Nat Immunol, 3 (10), pp. 940-943. | Show Abstract | Read more

The multiple sclerosis (MS)-associated HLA major histocompatibility complex (MHC) class II alleles DRB1*1501, DRB5*0101 and DQB1*0602 are in strong linkage disequilibrium, making it difficult to determine which is the principal MS risk gene. Here we show that together the DRB1 and DRB5 loci may influence susceptibility to MS. We demonstrate that a T cell receptor (TCR) from an MS patient recognized both a DRB1*1501-restricted myelin basic protein (MBP) and DRB5*0101-restricted Epstein-Barr virus (EBV) peptide. Crystal structure determination of the DRB5*0101-EBV peptide complex revealed a marked degree of structural equivalence to the DRB1*1501-MBP peptide complex at the surface presented for TCR recognition. This provides structural evidence for molecular mimicry involving HLA molecules. The structural details suggest an explanation for the preponderance of MHC class II associations in HLA-associated diseases.

Williams CJ, Zhang Y, Timms A, Bonavita G, Caeiro F, Broxholme J, Cuthbertson J, Jones Y, Marchegiani R, Reginato A et al. 2002. Autosomal dominant familial calcium pyrophosphate dihydrate deposition disease is caused by mutation in the transmembrane protein ANKH. Am J Hum Genet, 71 (4), pp. 985-991. | Show Abstract | Read more

Familial autosomal dominant calcium pyrophosphate dihydrate (CPPD) chondrocalcinosis has previously been mapped to chromosome 5p15. We have identified a mutation in the ANKH gene that segregates with the disease in a family with this condition. ANKH encodes a putative transmembrane inorganic pyrophosphate (PPi) transport channel. We postulate that loss of function of ANKH causes elevated extracellular PPi levels, predisposing to CPPD crystal deposition.

Gadola SD, Zaccai NR, Harlos K, Shepherd D, Castro-Palomino JC, Ritter G, Schmidt RR, Jones EY, Cerundolo V. 2002. Structure of human CD1b with bound ligands at 2.3 A, a maze for alkyl chains. Nat Immunol, 3 (8), pp. 721-726. | Show Abstract | Read more

The human genome encodes five nonpolymorphic major histocompatibility complex class I-like glycoproteins, CD1a to CD1e, that present lipid antigens for specific recognition by T lymphocytes. Using single alkyl chain detergents, we developed a protocol to generate recombinant human CD1b-lipid complexes. We present here the crystal structures of CD1b in complex with either phosphatidylinositol or ganglioside GM2 at 2.3 A and 2.8 A resolutions, respectively. The antigen-binding groove houses four interlinked hydrophobic channels that are occupied by the alkyl chains of the glycolipid plus two detergent molecules. A distinct exit beneath the alpha 2 helix further contributes to the plasticity of the binding groove. These structures reveal the mechanism by which two alkyl chain lipids bind to CD1b, and how CD1b can adapt to ligands of different alkyl chain length. They also suggest how very long alkyl chains, such as those of mycolic acid, could be fully contained within the binding groove. These results extend the spectrum of potential CD1b ligands by revealing that, in addition to two alkyl chain lipids, mono-alkyl and triple-alkyl chain lipids can be accommodated in the binding groove.

Hon WC, Wilson MI, Harlos K, Claridge TD, Schofield CJ, Pugh CW, Maxwell PH, Ratcliffe PJ, Stuart DI, Jones EY. 2002. Structural basis for the recognition of hydroxyproline in HIF-1 alpha by pVHL. Nature, 417 (6892), pp. 975-978. | Show Abstract | Read more

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that controls cellular and systemic homeostatic responses to oxygen availability. HIF-1 alpha is the oxygen-regulated subunit of HIF-1, an alpha beta heterodimeric complex. HIF-1 alpha is stable in hypoxia, but in the presence of oxygen it is targeted for proteasomal degradation by the ubiquitination complex pVHL, the protein of the von Hippel Lindau (VHL) tumour suppressor gene and a component of an E3 ubiquitin ligase complex. Capture of HIF-1 alpha by pVHL is regulated by hydroxylation of specific prolyl residues in two functionally independent regions of HIF-1 alpha. The crystal structure of a hydroxylated HIF-1 alpha peptide bound to VCB (pVHL, elongins C and B) and solution binding assays reveal a single, conserved hydroxyproline-binding pocket in pVHL. Optimized hydrogen bonding to the buried hydroxyprolyl group confers precise discrimination between hydroxylated and unmodified prolyl residues. This mechanism provides a new focus for development of therapeutic agents to modulate cellular responses to hypoxia.

Brown J, Esnouf RM, Jones MA, Linnell J, Harlos K, Hassan AB, Jones EY. 2002. Structure of a functional IGF2R fragment determined from the anomalous scattering of sulfur. EMBO J, 21 (5), pp. 1054-1062. | Show Abstract | Read more

Insulin-like growth factor II receptor (IGF2R) is a multifunctional cell surface receptor implicated in tumour suppression. Its growth inhibitory activity has been associated with an ability to bind IGF-II. IGF2R contains 15 homologous extracellular domains, with domain 11 primarily responsible for IGF-II binding. We report a 1.4 A resolution crystal structure of domain 11, solved using the anomalous scattering signal of sulfur. The structure consists of two crossed beta-sheets forming a flattened beta-barrel. Structural analysis identifies the putative IGF-II binding site at one end of the beta-barrel whilst crystal lattice contacts suggest a model for the full-length IGF2R extracellular region. The structure factors and coordinates of IGF2R domain 11 have been deposited in the Protein Data Bank (accession codes 1GP0 and 1GP3).

Boyton RJ, Zaccai N, Jones EY, Altmann DM. 2002. CD4 T cells selected by antigen under Th2 polarizing conditions favor an elongated TCR alpha chain complementarity-determining region 3. J Immunol, 168 (3), pp. 1018-1027. | Show Abstract

The affinity of the MHC/peptide/TCR interaction is thought to be one factor determining the differentiation of CD4+ T cells into Th1 or Th2 phenotypes. To study whether CD4+ cells generated under conditions favoring Th1 or Th2 responses select structurally different TCRs, Th1 and Th2 clones and lines were generated from nonobese diabetic and nonobese diabetic H2-E transgenic mice against the peptides proteolipoprotein 56-70, glutamic acid decarboxylase(65) 524-543, and heat shock protein-60 peptides 168-186 and 248-264. Th1/Th2 polarization allowed the generation of clones and lines with fixed peptide specificity and class II restriction but differing in Th1/Th2 phenotype in which the impact on TCR selection and structure could be studied. The Th2 clones tended to use longer TCR complementarity-determining region (CDR)3alpha loops than their Th1 counterparts. This trend was confirmed by analyzing TCRalpha transcripts from Th1 and Th2 polarized, bulk populations. Molecular modeling of Th1- and Th2-derived TCRs demonstrated that Th2 CDR3alpha comprised larger side chain residues than Th1 TCRs. The elongated, bulky Th2 CDR3alpha loops may be accommodated at the expense of less optimal interactions between the MHC class II/peptide and other CDR loops of the TCR. We propose that CD4+ T cells selected from the available repertoire under Th2 polarizing conditions tend to have elongated TCR CDR3alpha loops predicted to alter TCR binding, reducing contact at other interfaces and potentially leading to impeded TCR triggering.

Maenaka K, van der Merwe PA, Stuart DI, Jones EY, Sondermann P. 2001. The human low affinity Fcgamma receptors IIa, IIb, and III bind IgG with fast kinetics and distinct thermodynamic properties. J Biol Chem, 276 (48), pp. 44898-44904. | Show Abstract | Read more

Fcgamma receptors (FcgammaRs) are expressed on all immunologically active cells. They bind the Fc portion of IgG, thereby triggering a range of immunological functions. We have used surface plasmon resonance to analyze the kinetic and thermodynamic properties of the interactions between the ectodomains of human low affinity FcgammaRs (FcgammaRIIa, FcgammaRIIb, and FcgammaRIIIb-NA2) and IgG1 or the Fc fragment of IgG1. All three receptors bind Fc or IgG with similarly low affinities (K(D) approximately 0.6-2.5 microm) and fast kinetics, suggesting that FcgammaR-mediated recognition of aggregated IgG and IgG-coated particles or cells is mechanistically similar to cell-cell recognition. Interestingly, the Fc receptors exhibit distinct thermodynamic properties. Whereas the binding of the FcgammaRIIa and FcgammaRIIb to Fc is driven by favorable entropic and enthalpic changes, the binding of FcgammaRIII is characterized by highly unfavorable entropic changes. Although the structural bases for these differences remain to be determined, they suggest that the molecular events coupled to the binding differ among the low affinity FcgammaRs.

Walley AJ, Chavanas S, Moffatt MF, Esnouf RM, Ubhi B, Lawrence R, Wong K, Abecasis GR, Jones EY, Harper JI et al. 2001. Gene polymorphism in Netherton and common atopic disease. Nat Genet, 29 (2), pp. 175-178. | Show Abstract | Read more

Atopic dermatitis (AD) and asthma are characterized by IgE-mediated atopic (allergic) responses to common proteins (allergens), many of which are proteinases. Loci influencing atopy have been localized to a number of chromosomal regions, including the chromosome 5q31 cytokine cluster. Netherton disease is a rare recessive skin disorder in which atopy is a universal accompaniment. The gene underlying Netherton disease (SPINK5) encodes a 15-domain serine proteinase inhibitor (LEKTI) which is expressed in epithelial and mucosal surfaces and in the thymus. We have identified six coding polymorphisms in SPINK5 (Table 1) and found that a Glu420-->Lys variant shows significant association with atopy and AD in two independent panels of families. Our results implicate a previously unrecognized pathway for the development of common allergic illnesses.

Dorrell L, Willcox BE, Jones EY, Gillespie G, Njai H, Sabally S, Jaye A, DeGleria K, Rostron T, Lepin E et al. 2001. Cytotoxic T lymphocytes recognize structurally diverse, clade-specific and cross-reactive peptides in human immunodeficiency virus type-1 gag through HLA-B53. Eur J Immunol, 31 (6), pp. 1747-1756. | Show Abstract | Read more

Human immunodeficiency virus type-1 (HIV-1) cytotoxic T lymphocyte (CTL) epitopes have largely been defined in Caucasian populations infected with clade B virus. Identification of potentially protective CTL epitopes in non-B clade-infected African subjects is important for vaccine development. In a study of CTL responses in clade A-infected Gambians, using cytotoxicity, interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISpot) and HLA-B53-peptide tetramer assays, we identified three HLA-B53-restricted epitopes in HIV-1 gag p24. CTL specific for an epitope in a highly immunogenic region of the p24 protein showed no cross-reactivity to other HIV-1 clades. Two of the epitopes would not have been predicted from the peptide-binding motif due to the absence of a proline anchor at position 2. Structural analysis of HLA-B53 and its relative, HLA B35, enabled us to re-define the peptide-binding motif to include other P2 anchors. These results demonstrate the value of combined immunological and structural analyses in defining novel CTL epitopes and have implications for HIV-1 vaccine design.

Jones EY. 2001. Blueprints for life or death. Nat Immunol, 2 (5), pp. 379-380. | Show Abstract | Read more

NK cell receptors either activate or inhibit the fratricidal tendencies of NK cells. Structural analysis of receptor-ligand complexes of both types of receptors reveals striking similarities in form, despite the diverse function.

Davis SJ, Ikemizu S, Collins AV, Fennelly JA, Harlos K, Jones EY, Stuart DI. 2001. Crystallization and functional analysis of a soluble deglycosylated form of the human costimulatory molecule B7-1. Acta Crystallogr D Biol Crystallogr, 57 (Pt 4), pp. 605-608. | Show Abstract | Read more

The interactions of B7-1 with CD28 and CTLA-4 modulate the course of human immune responses, making B7-1 an important target for developing structure-based therapeutics. B7-1 is, however, one of the most heavily glycosylated proteins found at the leukocyte cell surface, complicating the structural analysis of this molecule. Methods for the production, crystallization and selenomethionine labelling of a soluble deglycosylated form of this molecule are described. The protein readily forms both tetragonal plate and bipyramidal crystals belonging to space groups I4(1)22, with unit-cell parameters a = b = 56.9, c = 298.7 A, and P4(1)22 (or P4(3)22), with unit-cell parameters a = b = 89.0, c = 261.9 A, respectively. The I4(1)22 and primitive crystal forms diffract to 2.7 and 3.5 A, respectively. Surface plasmon resonance-based assays indicate that the ligand-binding properties of sB7-1 are unaffected by deglycosylation. Since none of the methods relied on any special structural properties of sB7-1, it is proposed that this novel combination of procedures could in principle be adapted to the systematic analysis of many other glycoproteins of structural or functional interest.

Tafuro S, Meier UC, Dunbar PR, Jones EY, Layton GT, Hunter MG, Bell JI, McMichael AJ. 2001. Reconstitution of antigen presentation in HLA class I-negative cancer cells with peptide-beta2m fusion molecules. Eur J Immunol, 31 (2), pp. 440-449. | Show Abstract | Read more

Engineered MHC-peptide targets capable of inducing recognition by CTL may prove useful in designing vaccines for infectious disease and cancer. We tested whether peptides directly linked to beta2-microglobulin (beta2m) could complex with human HLA class I heavy chain, and could be recognized by human CTL, both as soluble reagents and as cell surface constituents. An HLA-A2-restricted peptide epitope was physically linked to the N terminus of human beta2m. This fusion protein refolded efficiently in vitro with HLA-A2 heavy chain, and when multimerized, the resultant complexes ("fusamers") bound specifically to appropriate CTL clones. These fused peptide/MHC complexes were as efficient as standard tetrameric peptide/MHC complexes in recognizing antigen-specific CTL. When the fusion protein was delivered to target cells using a retroviral vector, these cells were recognized and killed by appropriate CTL clones. Efficient sensitization to CTL lysis was achieved in TAP-negative and beta2m-negative cell lines, as well as in unmutated B cell lines, proving that such constructs may be effective in inducing CTL even when the MHC class I pathway has been disrupted. Specific peptides covalently linked to beta2m and delivered via retroviral vectors may be useful reagents for in vivo priming of CTL against epitopes of clinical relevance.

Jones EY. 2000. The tumour necrosis factor receptor family: life or death choices. Curr Opin Struct Biol, 10 (6), pp. 644-648. | Show Abstract | Read more

The past twelve months have seen a renewal of interest in the therapeutic potential of members of the tumour necrosis factor receptor family and their cytokine ligands. This biomedical interest has spawned a number of structural studies, which have significantly deepened our understanding of the molecular basis for the function of these cell-surface signalling systems. The fresh data have revealed unexpected mechanisms conferring ligand-receptor specificity and have highlighted the structural requirements for the initiation of intracellular signalling.

Maenaka K, Maenaka T, Tomiyama H, Takiguchi M, Stuart DI, Jones EY. 2000. Nonstandard peptide binding revealed by crystal structures of HLA-B*5101 complexed with HIV immunodominant epitopes. J Immunol, 165 (6), pp. 3260-3267. | Show Abstract

The crystal structures of the human MHC class I allele HLA-B*5101 in complex with 8-mer, TAFTIPSI, and 9-mer, LPPVVAKEI, immunodominant peptide epitopes from HIV-1 have been determined by x-ray crystallography. In both complexes, the hydrogen-bonding network in the N-terminal anchor (P1) pocket is rearranged as a result of the replacement of the standard tyrosine with histidine at position 171. This results in a nonstandard positioning of the peptide N terminus, which is recognized by B*5101-restricted T cell clones. Unexpectedly, the P5 peptide residues appear to act as anchors, drawing the peptides unusually deeply into the peptide-binding groove of B51. The unique characteristics of P1 and P5 are likely to be responsible for the zig-zag conformation of the 9-mer peptide and the slow assembly of B*5101. A comparison of the surface characteristics in the alpha1-helix C-terminal region for B51 and other MHC class I alleles highlights mainly electrostatic differences that may be important in determining the specificity of human killer cell Ig-like receptor binding.

Deller MC, Hudson KR, Ikemizu S, Bravo J, Jones EY, Heath JK. 2000. Crystal structure and functional dissection of the cytostatic cytokine oncostatin M. Structure, 8 (8), pp. 863-874. | Show Abstract | Read more

BACKGROUND: The cytokine oncostatin M (OSM) inhibits growth of certain tumour-derived cells, induces proliferation in other cell types (e.g. haemangioblasts) and is a mediator of inflammatory responses. Its mechanism of action is via specific binding to gp130 and either the leukaemia inhibitory factor receptor (LIFR) or oncostatin M receptor (OSMR) systems at the cell surface to form an active signalling complex. RESULTS: We report here the crystal structure of human oncostatin M (hOSM) along with mutagenesis data which map the receptor-binding epitopes of the molecule. The structure was determined to a resolution of 2.2 A and conforms to the haematopoietin cytokine up-up-down-down four-helix bundle topology. The site 2 epitope, responsible for gp130 binding, is centred around Gly120 which forms a 'dimple' on the surface of the molecule located on helices A and C. The site 3 motif, responsible for LIFR and OSMR binding, consists of a protruding Phe160/Lys163 pair located at the start of helix D. CONCLUSIONS: The data presented allow functional dissection of the receptor-binding interfaces to atomic resolution. Modelling suggests that the gp130 residue Phe169 packs into the site 2 dimple in an analogous fashion to structurally equivalent residues at the growth hormone-growth hormone receptor interface, implying that certain key features may underlie recognition across the whole cytokine/receptor superfamily. Conversely, detailed comparison of the available structures suggests that variations on a common theme dictate the specificity of receptor-ligand interactions within the gp130 family of cytokines.

Krausa P, Münz C, Keilholz W, Stevanovic S, Jones EY, Browning M, Bunce M, Rammensee HG, McMichael A. 2000. Definition of peptide binding motifs amongst the HLA-A*30 allelic group. Tissue Antigens, 56 (1), pp. 10-18. | Show Abstract | Read more

HLA class I molecules present endogenously processed peptide ligands for surveillance by the T-cell receptor. This potentially immunogenic surface of HLA and peptide is a consequence of the polymorphism found within the HLA molecule and its preference for ligand binding together with peptide conformation within the binding groove. To investigate the relation between the polymorphic differences between some closely related HLA alleles and their effect on peptide preference, transfectants were established, each containing one of four allelic variants of HLA-A*30. Peptides from all four transfectants were eluted, and both individual ligands and peptide pools were sequenced. The data shows two distinct peptide motifs which distinguish A*3001 from the other three known A*30 variants. Differences in preferences at minor positions within the peptide sequence were noted between A*3002, A*3003 and A*3004, providing additional evidence of the implications of sequence polymorphism to HLA function.

Gao GF, Willcox BE, Wyer JR, Boulter JM, O'Callaghan CA, Maenaka K, Stuart DI, Jones EY, Van Der Merwe PA, Bell JI, Jakobsen BK. 2000. Classical and nonclassical class I major histocompatibility complex molecules exhibit subtle conformational differences that affect binding to CD8alphaalpha. J Biol Chem, 275 (20), pp. 15232-15238. | Show Abstract | Read more

The cell surface molecules CD4 and CD8 greatly enhance the sensitivity of T-cell antigen recognition, acting as "co-receptors" by binding to the same major histocompatibility complex (MHC) molecules as the T-cell receptor (TCR). Here we use surface plasmon resonance to study the binding of CD8alphaalpha to class I MHC molecules. CD8alphaalpha bound the classical MHC molecules HLA-A*0201, -A*1101, -B*3501, and -C*0702 with dissociation constants (K(d)) of 90-220 microm, a range of affinities distinctly lower than that of TCR/peptide-MHC interaction. We suggest such affinities apply to most CD8alphaalpha/classical class I MHC interactions and may be optimal for T-cell recognition. In contrast, CD8alphaalpha bound both HLA-A*6801 and B*4801 with a significantly lower affinity (>/=1 mm), consistent with the finding that interactions with these alleles are unable to mediate cell-cell adhesion. Interestingly, CD8alphaalpha bound normally to the nonclassical MHC molecule HLA-G (K(d) approximately 150 microm), but only weakly to the natural killer cell receptor ligand HLA-E (K(d) >/= 1 mm). Site-directed mutagenesis experiments revealed that variation in CD8alphaalpha binding affinity can be explained by amino acid differences within the alpha3 domain. Taken together with crystallographic studies, these results indicate that subtle conformational changes in the solvent exposed alpha3 domain loop (residues 223-229) can account for the differential ability of both classical and nonclassical class I MHC molecules to bind CD8.

Kasper C, Rasmussen H, Kastrup JS, Ikemizu S, Jones EY, Berezin V, Bock E, Larsen IK. 2000. Structural basis of cell-cell adhesion by NCAM. Nat Struct Biol, 7 (5), pp. 389-393. | Show Abstract | Read more

The neural cell adhesion molecule NCAM, a member of the immunoglobulin superfamily, mediates cell-cell recognition and adhesion via a homophilic interaction. NCAM plays a key role during development and regeneration of the nervous system and is involved in synaptic plasticity associated with memory and learning. The 1.85 A crystal structure of the two N-terminal extracellular domains of NCAM reported here provides a structural basis for the homophilic interaction. The molecular packing of the two-domain structure reveals a cross shaped antiparallel dimer, and provides fundamental insight into trans-cellular recognition mediated by NCAM.

Deller MC, Yvonne Jones E. 2000. Cell surface receptors. Curr Opin Struct Biol, 10 (2), pp. 213-219. | Show Abstract | Read more

During the past year, the database of ligand-receptor complexes has essentially doubled. These new results have immeasurably extended and expanded our view of cell surface receptor structure and function. The flood of data has revealed new models for receptor cross-linking, demonstrating the potential stringency of the extracellular requirements for the initiation of intracellular signalling and highlighting unexpected interactions suggestive of higher order clustering.

Ikemizu S, Gilbert RJ, Fennelly JA, Collins AV, Harlos K, Jones EY, Stuart DI, Davis SJ. 2000. Structure and dimerization of a soluble form of B7-1. Immunity, 12 (1), pp. 51-60. | Show Abstract | Read more

B7-1 (CD80) and B7-2 (CD86) are glycoproteins expressed on antigen-presenting cells. The binding of these molecules to the T cell homodimers CD28 and CTLA-4 (CD152) generates costimulatory and inhibitory signals in T cells, respectively. The crystal structure of the extracellular region of B7-1 (sB7-1), solved to 3 A resolution, consists of a novel combination of two Ig-like domains, one characteristic of adhesion molecules and the other previously seen only in antigen receptors. In the crystal lattice, sB7-1 unexpectedly forms parallel, 2-fold rotationally symmetric homodimers. Analytical ultracentrifugation reveals that sB7-1 also dimerizes in solution. The structural data suggest a mechanism whereby the avidity-enhanced binding of B7-1 and CTLA-4 homodimers, along with the relatively high affinity of these interactions, favors the formation of very stable inhibitory signaling complexes.

Maenaka K, Jones EY. 1999. MHC superfamily structure and the immune system. Curr Opin Struct Biol, 9 (6), pp. 745-753. | Show Abstract | Read more

During the past year, a plethora of structural information has provided detailed insights into the interactions between classical MHC class I molecules and their cognate receptors on T cells. Likewise, there have been major advances in our knowledge of the structures and functions of five nonclassical MHC-like molecules: HLA-DM (murine H2-M), HLA-E, HFE, ZAG and MIC-A.

Robinson RC, Radziejewski C, Spraggon G, Greenwald J, Kostura MR, Burtnick LD, Stuart DI, Choe S, Jones EY. 1999. The structures of the neurotrophin 4 homodimer and the brain-derived neurotrophic factor/neurotrophin 4 heterodimer reveal a common Trk-binding site. Protein Sci, 8 (12), pp. 2589-2597. | Show Abstract | Read more

The neurotrophins are growth factors that are involved in the development and survival of neurons. Neurotrophin release by a target tissue results in neuron growth along the neurotrophin concentration gradient, culminating in the eventual innervation of the target tissue. These activities are mediated through trk cell surface receptors. We have determined the structures of the heterodimer formed between brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4), as well as the structure of homodimer of NT4. We also present the structure of the Neurotrophin 3 homodimer, which is refined to higher resolution than previously published. These structures provide the first views of the architecture of the NT4 protomer. Comparison of the surface of a model of the BDNF homodimer with the structures of the neurotrophin homodimers reveals common features that may be important in the binding between the neurotrophins and their receptors. In particular, there exists an analogous region on the surface of each neurotrophin that is likely to be involved in trk receptor binding. Variations in sequence on the periphery of this common region serve to confer trk receptor specificity.

Willcox BE, Gao GF, Wyer JR, O'Callaghan CA, Boulter JM, Jones EY, van der Merwe PA, Bell JI, Jakobsen BK. 1999. Production of soluble alphabeta T-cell receptor heterodimers suitable for biophysical analysis of ligand binding. Protein Sci, 8 (11), pp. 2418-2423. | Show Abstract | Read more

A method to produce alphabeta T-cell receptors (TCRs) in a soluble form suitable for biophysical analysis was devised involving in vitro refolding of a TCR fusion protein. Polypeptides corresponding to the variable and constant domains of each chain of a human and a murine receptor, fused to a coiled coil heterodimerization motif from either c-Jun (alpha) or v-Fos (beta), were overexpressed separately in Escherichia coli. Following recovery from inclusion bodies, the two chains of each receptor were denatured, and then refolded together in the presence of denaturants. For the human receptor, which is specific for the immunodominant influenza A HLA-A2-restricted matrix epitope (M58-66), a heterodimeric protein was purified in milligram yields and found to be homogeneous, monomeric, antibody-reactive, and stable at concentrations lower than 1 microM. Using similar procedures, analogous results were obtained with a murine receptor specific for an influenza nucleoprotein epitope (366-374) restricted by H2-Db. Production of these receptors has facilitated a detailed analysis of viral peptide-Major Histocompatibility Complex (peptide-MHC) engagement by the TCR using both surface plasmon resonance (SPR) and, in the case of the human TCR, isothermal titration calorimetry (ITC) (Willcox et al., 1999). The recombinant methods described should enable a wide range of TCR-peptide-MHC interactions to be studied and may also have implications for the production of other heterodimeric receptor molecules.

Mongkolsapaya J, Grimes JM, Chen N, Xu XN, Stuart DI, Jones EY, Screaton GR. 1999. Structure of the TRAIL-DR5 complex reveals mechanisms conferring specificity in apoptotic initiation. Nat Struct Biol, 6 (11), pp. 1048-1053. | Show Abstract | Read more

TRAIL, an apoptosis inducing ligand, has at least four cell surface receptors including the death receptor DR5. Here we report the crystal structure at 2.2 A resolution of a complex between TRAIL and the extracellular region of DR5. TRAIL forms a central homotrimer around which three DR5 molecules bind. Radical differences in the surface charge of the ligand, together with variation in the alignment of the two receptor domains confer specificity between members of these ligand and receptor families. The existence of a switch mechanism allowing variation in receptor domain alignment may mean that it is possible to engineer receptors with multiple specificities by exploiting contact positions unique to individual receptor-ligand pairs.

Bowness P, Zaccai N, Bird L, Jones EY. 1999. HLA-B27 and disease pathogenesis: new structural and functional insights. Expert Rev Mol Med, 1999 (16), pp. 1-10. | Show Abstract | Read more

The human leukocyte antigen class I allele HLA-B27 is a major histocompatibility complex (MHC) antigen that is strongly associated with the spondyloarthritic group of human rheumatic diseases, the most common of which is ankylosing spondylitis. Although the mechanism underlying this disease association remains unknown, numerous theories have been proposed. Much more is known of the natural role of HLA-B27 in binding and presenting antigenic peptides to T cells. The 'arthritogenic peptide hypothesis' suggests that the role of HLA-B27 in disease relates to its specificity for binding certain peptides. Recently, it has also been shown that HLA-B27 has an unusual cell biology and can adopt a novel homodimeric structure. In this review, a molecular model of the HLA-B27 homodimer is presented and the possible pathogenic significance of such a structure is discussed.

Maenaka K, Juji T, Nakayama T, Wyer JR, Gao GF, Maenaka T, Zaccai NR, Kikuchi A, Yabe T, Tokunaga K et al. 1999. Killer cell immunoglobulin receptors and T cell receptors bind peptide-major histocompatibility complex class I with distinct thermodynamic and kinetic properties. J Biol Chem, 274 (40), pp. 28329-28334. | Show Abstract | Read more

Human natural killer cells and a subset of T cells express a repertoire of killer cell immunoglobulin receptors (KIRs) that recognize major histocompatibility complex (MHC) class I molecules. KIRs and T cell receptors (TCRs) bind in a peptide-dependent manner to overlapping regions of peptide-MHC class I complexes. KIRs with two immunoglobulin domains (KIR2Ds) recognize distinct subsets of HLA-C alleles. Here we use surface plasmon resonance to study the binding of soluble forms of KIR2DL1 and KIR2DL3 to several peptide-HLA-Cw7 complexes. KIR2DL3 bound to the HLA-Cw7 allele presenting the peptide RYRPGTVAL with a 1:1 stoichiometry and an affinity (K(d) approximately 7 microM at 25 degrees C) within the range of values measured for other cell-cell recognition molecules, including the TCR. Although KIR2DL1 is reported not to recognize the HLA-Cw7 allele in functional assays, it bound RYRPGTVAL/HLA-Cw7, albeit with a 10-20-fold lower affinity. TCR/peptide-MHC interactions are characterized by comparatively slow kinetics and unfavorable entropic changes (Willcox, B. E., Gao, G. F., Wyer, J. R. , Ladbury, J. E., Bell, J. I., Jakobsen, B. K., and van der Merwe, P. A. (1999) Immunity 10, 357-365), suggesting that binding is accompanied by conformational adjustments. In contrast, we show that KIR2DL3 binds RYRPGTVAL/HLA-Cw7 with fast kinetics and a favorable binding entropy, consistent with rigid body association. These results indicate that KIR/peptide-MHC class I interactions have properties typical of other cell-cell recognition molecules, and they highlight the unusual nature of TCR/peptide-MHC recognition.

Butters TD, Sparks LM, Harlos K, Ikemizu S, Stuart DI, Jones EY, Davis SJ. 1999. Effects of N-butyldeoxynojirimycin and the Lec3.2.8.1 mutant phenotype on N-glycan processing in Chinese hamster ovary cells: application to glycoprotein crystallization. Protein Sci, 8 (8), pp. 1696-1701. | Show Abstract | Read more

Heterologous gene expression in either (1) the glycosylation-defective, mutant Chinese hamster ovary cell line, Lec3.2.8.1, or (2) the presence of the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin facilitates the trimming of N-linked glycans of glycoproteins to single N-acetylglucosamine (GlcNAc) residues with endoglycosidase H (endo H). Both approaches are somewhat inefficient, however, with as little as 12% of the total protein being rendered fully endo H-sensitive under these conditions. It is shown here that the combined effects of these approaches on the restriction of oligosaccharide processing are essentially additive, thereby allowing the production of glycoproteins that are essentially completely endo H-sensitive. The preparation of a soluble chimeric form of CD58, the ligand of the human T-cell surface recognition molecule CD2, illustrates the usefulness of the combined approach when expression levels are low or the deglycosylated protein is unstable at low pH. The endo H-treated chimera produced crystals of space group P3(1)21 or P3(2)21, and unit cell dimensions a = b = 116.4 A, c = 51.4 A alpha = beta = 90 degrees , gamma = 120 degrees , that diffract to a maximum resolution of 1.8 A.

Maenaka K, Juji T, Stuart DI, Jones EY. 1999. Crystal structure of the human p58 killer cell inhibitory receptor (KIR2DL3) specific for HLA-Cw3-related MHC class I. Structure, 7 (4), pp. 391-398. | Show Abstract | Read more

BACKGROUND: T cells and natural killer (NK) cells perform complementary roles in the cellular immune system. T cells identify infected cells directly through recognition of antigenic peptides that are displayed at the target cell surface by the classical major histocompatibility complex (MHC) class I molecules. NK cells monitor the target cell surface for malfunction of this display system, lysing potentially infected cells that might otherwise evade recognition by the T cells. Human killer cell inhibitory receptors (KIRs) control this process by either inhibiting or activating the cytotoxic activity of NK cells via specific binding to MHC class I molecules on the target cell. RESULTS: We report the crystal structure of the extracellular region of the human p58 KIR (KIR2DL3), which is specific for the human MHC class I molecule HLA-Cw3 and related alleles. The structure shows the predicted topology of two tandem immunoglobulin-like domains, but comparison with the previously reported structure of the related receptor KIR2DL1 reveals an unexpected change of 23 degrees in the relative orientation of these domains. CONCLUSIONS: The altered orientation of the immunoglobulin-like domains maintains an unusually acute interdomain elbow angle, which therefore appears to be a distinctive feature of the KIRs. The putative MHC class I binding site is located on the outer surface of the elbow, spanning both domains. The unexpected observation that this binding site can be modulated by differences in the relative domain orientations has implications for the general mechanism of KIR-MHC class I complex formation.

Ikemizu S, Sparks LM, van der Merwe PA, Harlos K, Stuart DI, Jones EY, Davis SJ. 1999. Crystal structure of the CD2-binding domain of CD58 (lymphocyte function-associated antigen 3) at 1.8-A resolution. Proc Natl Acad Sci U S A, 96 (8), pp. 4289-4294. | Show Abstract | Read more

The binding of the cell surface molecule CD58 (formerly lymphocyte function-associated antigen 3) to its ligand, CD2, significantly increases the sensitivity of antigen recognition by T cells. This was the first heterophilic cell adhesion interaction to be discovered and is now an important paradigm for analyzing the structural basis of cell-cell recognition. The crystal structure of a CD2-binding chimeric form of CD58, solved to 1.8-A resolution, reveals that the ligand binding domain of CD58 has the expected Ig superfamily V-set topology and shares several of the hitherto unique structural features of CD2, consistent with previous speculation that the genes encoding these molecules arose via duplication of a common precursor. Nevertheless, evidence for considerable divergence of CD2 and CD58 is also implicit in the structures. Mutations that disrupt CD2 binding map to the highly acidic surface of the AGFCC'C" beta-sheet of CD58, which, unexpectedly, lacks marked shape complementarity to the equivalent, rather more basic CD58-binding face of human CD2. The specificity of the very weak interactions of proteins mediating cell-cell recognition may often derive largely from electrostatic complementarity, with shape matching at the protein-protein interface being less exact than for interactions that combine specificity with high affinity, such as those involving antibodies.

Sharma A, Askari JA, Humphries MJ, Jones EY, Stuart DI. 1999. Crystal structure of a heparin- and integrin-binding segment of human fibronectin. EMBO J, 18 (6), pp. 1468-1479. | Show Abstract | Read more

The crystal structure of human fibronectin (FN) type III repeats 12-14 reveals the primary heparin-binding site, a clump of positively charged residues in FN13, and a putative minor site approximately 60 A away in FN14. The IDAPS motif implicated in integrin alpha4beta1 binding is at the FN13-14 junction, rendering the critical Asp184 inaccessible to integrin. Asp184 clamps the BC loop of FN14, whose sequence (PRARI) is reminiscent of the synergy sequence (PHSRN) of FN9. Mutagenesis studies prompted by this observation reveal that both arginines of the PRARI sequence are important for alpha4beta1 binding to FN12-14. The PRARI motif may represent a new class of integrin-binding sites. The spatial organization of the binding sites suggests that heparin and integrin may bind in concert.

Glithero A, Tormo J, Haurum JS, Arsequell G, Valencia G, Edwards J, Springer S, Townsend A, Pao YL, Wormald M et al. 1999. Crystal structures of two H-2Db/glycopeptide complexes suggest a molecular basis for CTL cross-reactivity. Immunity, 10 (1), pp. 63-74. | Show Abstract | Read more

Two synthetic O-GlcNAc-bearing peptides that elicit H-2Db-restricted glycopeptide-specific cytotoxic T cells (CTL) have been shown to display nonreciprocal patterns of cross-reactivity. Here, we present the crystal structures of the H-2Db glycopeptide complexes to 2.85 A resolution or better. In both cases, the glycan is solvent exposed and available for direct recognition by the T cell receptor (TCR). We have modeled the complex formed between the MHC-glycopeptide complexes and their respective TCRs, showing that a single saccharide residue can be accommodated in the standard TCR-MHC geometry. The models also reveal a possible molecular basis for the observed cross-reactivity patterns of the CTL clones, which appear to be influenced by the length of the CDR3 loop and the nature of the immunizing ligand.

Esnouf RM, Ren J, Garman EF, Somers DO, Ross CK, Jones EY, Stammers DK, Stuart DI. 1998. Continuous and discontinuous changes in the unit cell of HIV-1 reverse transcriptase crystals on dehydration. Acta Crystallogr D Biol Crystallogr, 54 (Pt 5), pp. 938-953. | Show Abstract | Read more

A crystal form of HIV-1 reverse transcriptase (RT) complexed with inhibitors showed diffraction to a high-resolution limit of 3.7 A. Instability in the unit-cell dimensions of these crystals was observed during soaking experiments, but the range of this variability and consequent change in lattice order was revealed by a chance observation of dehydration. Deliberately induced dehydration results in crystals having a variety of unit cells, the best-ordered of which show diffraction to a minimum Bragg spacing of 2.2 A. In order to understand the molecular basis for this phenomenon, the initial observation of dehydration, the data sets from dehydrated crystals, the crystal packing and the domain conformation of RT are analysed in detail here. This analysis reveals that the crystals undergo remarkable changes following a variety of possible dehydration pathways: some changes occur gradually whilst others are abrupt and require significant domain rearrangements. Comparison of domain arrangements in different crystal forms gives insight into the flexibility of RT which, in turn, may reflect the internal motions allowing this therapeutically important enzyme to fulfill its biological function.

Bowness P, Allen RL, Barclay DN, Jones EY, McMichael AJ. 1998. Importance of a conserved TCR J alpha-encoded tyrosine for T cell recognition of an HLA B27/peptide complex. Eur J Immunol, 28 (9), pp. 2704-2713. | Show Abstract | Read more

Human HLA B27-restricted cytotoxic T lymphocytes (CTL) specific for the influenza A epitope NP383-391 use similar TCR alpha and beta chains, with two closely related J alpha segments used by six of nine CTL clones from three unrelated donors (Bowness et al., Eur J. Immunol. 1993. 23: 1417-1421). The role of TCR complementarity-determining region (CDR)3alpha residues 93 and 100-102 was examined by site-directed mutagenesis, following expression of the TCR alpha and beta extracellular domains from one clone as a TCR zeta fusion heterodimer in rat basophil leukemia (RBL) cells. For the first time we have measured direct binding of tetrameric HLA B*2705/NP383-391 complexes to transfected TCR. Independently peptide-pulsed antigen-presenting cells (APC) were used to induce TCR-mediated degranulation of RBL transfectants. Our results show a key role for the conserved TCRalpha CDR3 J alpha-encoded residue Y102 in recognition of HLA B27/NP383-391. Thus the Y102D mutation abolished both tetramer binding and degranulation in the presence of peptide-pulsed APC. Even the Y102F mutation, differing only by a single hydroxyl group from the native TCR, abolished detectable degranulation. Further mutations F93A and S100R also abolished recognition. Interestingly, the N101A mutation recognized HLA B27/NP in functional assays despite having significantly reduced tetramer binding, a finding consistent with "kinetic editing" models of T cell activation. Modeling of the GRb TCR CDR3alpha loop suggests that residue Y102 contacts the HLA B*2705 alpha1 helix. It is thus possible that selection of germ-line TCRAJ-encoded residues at position 102 may be MHC driven.

Ren J, Esnouf RM, Hopkins AL, Jones EY, Kirby I, Keeling J, Ross CK, Larder BA, Stuart DI, Stammers DK. 1998. 3'-Azido-3'-deoxythymidine drug resistance mutations in HIV-1 reverse transcriptase can induce long range conformational changes. Proc Natl Acad Sci U S A, 95 (16), pp. 9518-9523. | Show Abstract | Read more

HIV reverse transcriptase (RT) is one of the main targets for the action of anti-AIDS drugs. Many of these drugs [e.g., 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI)] are analogues of the nucleoside substrates used by the HIV RT. One of the main problems in anti-HIV therapy is the selection of a mutant virus with reduced drug sensitivity. Drug resistance in HIV is generated for nucleoside analogue inhibitors by mutations in HIV RT. However, most of these mutations are situated some distance from the polymerase active site, giving rise to questions concerning the mechanism of resistance. To understand the possible structural bases for this, the crystal structures of AZT- and ddI-resistant RTs have been determined. For the ddI-resistant RT with a mutation at residue 74, no significant conformational changes were observed for the p66 subunit. In contrast, for the AZT-resistant RT (RTMC) bearing four mutations, two of these (at 215 and 219) give rise to a conformational change that propagates to the active site aspartate residues. Thus, these drug resistance mutations produce an effect at the RT polymerase site mediated simply by the protein. It is likely that such long-range effects could represent a common mechanism for generating drug resistance in other systems.

Tanaka H, Walker RT, Hopkins AL, Ren J, Jones EY, Fujimoto K, Hayashi M, Miyasaka T, Baba M, Stammers DK, Stuart DI. 1998. Allosteric inhibitors against HIV-1 reverse transcriptase: design and synthesis of MKC-442 analogues having an omega-functionalized acyclic structure. Antivir Chem Chemother, 9 (4), pp. 325-332. | Show Abstract

Based on X-ray crystallographic analysis of MKC-442/human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) complex, analogues in which the N1-substituent is replaced with omega-functionalized alkyl groups were designed to improve the affinity for the enzyme. Synthesis of these compounds was carried out starting from MKC-442 by a sequence of reactions (N3-protection, removal of N1-ethoxymethyl group, alkylation, and N3-deprotection). The compounds were evaluated for anti-HIV activity. Structure-activity relationships are discussed in terms of the possible interaction with the enzyme.

Jones EY, Tormo J, Reid SW, Stuart DI. 1998. Recognition surfaces of MHC class I. Immunol Rev, 163 (1), pp. 121-128. | Show Abstract | Read more

Recent crystallographic results have provided close to atomic resolution views of the recognition events mediated by MHC class I molecules. The specificity-conferring interaction of MHC class I/peptide with a T-cell antigen receptor (TCR) appears dependent on certain key interactions with the MHC scaffold. These interactions, in particular those of the TCR V alpha domain, define a standard orientation for TCR binding. Previous studies on biologically significant variations in the TCR recognition surface presented by a series of MHC/variant peptide complexes can be reassessed in the light of this TCR-binding mode. The interaction of CD8 with MHC class I resembles that between antibody and antigen in the use of loops from the CD8 structure. The interaction is of very low affinity and buries equivalent surface area to that between the TCR and MHC class I but while the TCR/MHC interface shows poor surface shape complementarity the match in the conservative interaction between MHC and CD8 is precise.

Davis SJ, Davies EA, Tucknott MG, Jones EY, van der Merwe PA. 1998. The role of charged residues mediating low affinity protein-protein recognition at the cell surface by CD2. Proc Natl Acad Sci U S A, 95 (10), pp. 5490-5494. | Show Abstract | Read more

Insights into the structural basis of protein-protein recognition have come principally from the analysis of proteins such as antibodies, hormone receptors, and proteases that bind their ligands with relatively high affinity (Ka approximately 10(9) M-1). In contrast, few studies have been done on the very low affinity interactions mediating cell adhesion and cell-cell recognition. As a site of protein-protein recognition, the ligand binding face of the T lymphocyte cell-cell recognition molecule, CD2, which binds its ligands 10(4)- to 10(5)-fold more weakly than do antibodies and proteases, is unusual in being both very flat and highly charged. An analysis of the effect of mutations and ionic strength on CD2 binding to its ligand, CD48, indicates that these charged residues contribute little, if any, binding energy to this interaction. However, the loss of these charged residues is shown to markedly reduce ligand-binding specificity. Thus, the charged residues increase the specificity of CD2 binding without increasing the affinity. This phenomenon is likely to result from a requirement for electrostatic complementarity between charged binding surfaces to compensate for the removal, upon binding, of water interacting with the charged residues. It is proposed that this mode of recognition is highly suited to biological interactions requiring a low affinity because it uncouples increases in specificity from increases in affinity.

Gao GF, Gerth UC, Wyer JR, Willcox BE, O'Callaghan CA, Zhang Z, Jones EY, Bell JI, Jakobsen BK. 1998. Assembly and crystallization of the complex between the human T cell coreceptor CD8alpha homodimer and HLA-A2. Protein Sci, 7 (5), pp. 1245-1249. | Show Abstract | Read more

A strategy for overexpression in Escherichia coli of the extracellular immunoglobulin domain of human CD8alpha was devised using codon usage alterations in the 5' region of the gene, designed so as to prevent the formation of secondary structures in the mRNA. A fragment of CD8alpha, comprising residues 1-120 of the mature protein, excluding the signal peptide and the membrane-proximal stalk region, was recovered from bacterial inclusion bodies and refolded to produce a single species of homodimeric, soluble receptor. HLA-A2 heavy chain, beta2-microglobulin and a synthetic peptide antigen corresponding to the pol epitope from HIV-1 were also expressed in E. coli, refolded and purified. CD8alpha/HLA-A2 complexes were formed in solution and by co-crystallization with a stoichiometry of one CD8alpha alpha dimer to one HLA-A2-peptide unit.

Maenaka K, Juji T, Tadokoro K, Harlos K, Stuart DI, Jones EY. 1998. Crystallization and preliminary diffraction studies of the extracellular region of human p58 killer cell inhibitory receptor (KIR2). Acta Crystallogr D Biol Crystallogr, 54 (Pt 3), pp. 433-435. | Show Abstract | Read more

Molecules of the human killer cell inhibitory receptor (KIR) family, which belong to the immunoglobulin superfamily (IgSF), are expressed on the surface of natural killer (NK) cells and some subsets of T cells. These receptors function to mediate the inhibition or activation of cytotoxic activity by recognizing HLA class I molecules on the target cell. The extracellular region of a p58 KIR specific for HLA-Cw1,3,7 (KIR2) has been overproduced in Escherichia coli and purified. The recombinant KIR2 has been crystallized in 9-10% poly(ethylene glycol) methyl ether (average Mr = 8000), 50mM HEPES, 8% ethylene glycol, 0.5% octyl-beta-glucoside, pH 7.5, at 294 K using the sitting-drop vapour-diffusion method. Preliminary X-ray diffraction studies reveal the space group to be hexagonal (P6122 or P6522) with lattice constants a = b = 95.3, c = 130.8 A. A native data set (3 A resolution) has been collected at the Photon Factory (lambda = 1.0 A).

O'Callaghan CA, Tormo J, Willcox BE, Blundell CD, Jakobsen BK, Stuart DI, McMichael AJ, Bell JI, Jones EY. 1998. Production, crystallization, and preliminary X-ray analysis of the human MHC class Ib molecule HLA-E. Protein Sci, 7 (5), pp. 1264-1266. | Show Abstract | Read more

HLA-E is the first human class Ib major histocompatibility complex molecule to be crystallized. HLA-E is highly conserved and almost nonpolymorphic, and has recently been shown to be the first specialized ligand for natural killer cell receptors. In functional studies, HLA-E is unlike the class Ia MHC molecules in having tightly restricted peptide binding specificity. HLA-E binds a limited set of almost identical leader sequence peptides derived from class Ia molecules and presents these at the cell surface for recognition by natural killer cell receptors. We now show that the extracellular region of HLA-E forms a stable complex with beta2 microglobulin and can be refolded around synthetic peptide. Crystals of this complex formed slowly over four to six months in the presence of ammonium sulphate. The crystals diffract to 2.85 A with space group P3(1)21 and unit cell dimensions a = 182.2 A, b = 182.2 A, c = 88.4 A.

May AP, Robinson RC, Vinson M, Crocker PR, Jones EY. 1998. Crystal structure of the N-terminal domain of sialoadhesin in complex with 3' sialyllactose at 1.85 A resolution. Mol Cell, 1 (5), pp. 719-728. | Show Abstract | Read more

The structure of the functional N-terminal domain from the extracellular region of the cell surface receptor sialoadhesin has been determined in complex with the oligosaccharide 3' sialyllactose. This provides structural information for the siglec family of proteins. The structure conforms to the V-set immunoglobulin-like fold but contains several distinctive features, including an intra-beta sheet disulphide and a splitting of the standard beta strand G into two shorter strands. These novel features appear important in adapting the V-set fold for sialic acid-mediated recognition. Analysis of the complex with 3'sialyllactose highlights three residues, conserved throughout the siglec family, as key features of the sialic acid-binding template. The complex is representative of the functional recognition interaction with carbohydrate and as such provides detailed information for a heterotypic cell adhesion interaction.

Bravo J, Staunton D, Heath JK, Jones EY. 1998. Crystal structure of a cytokine-binding region of gp130. EMBO J, 17 (6), pp. 1665-1674. | Show Abstract | Read more

The structure of the cytokine-binding homology region of the cell surface receptor gp130 has been determined by X-ray crystallography at 2.0 A resolution. The beta sandwich structure of the two domains conforms to the topology of the cytokine receptor superfamily. This first structure of an uncomplexed receptor exhibits a similar L-shaped quaternary structure to that of ligand-bound family members and suggests a limited flexibility in relative domain orientation of some 3 degrees. The putative ligand-binding loops are relatively rigid, with a phenylalanine side chain similarly positioned to exposed aromatic residues implicated in ligand binding for other such receptors. The positioning and structure of the N-terminal portion of the polypeptide chain have implications for the structure and function of cytokine receptors, such as gp130, which contain an additional N-terminal immunoglobulin-like domain.

O'Callaghan CA, Tormo J, Willcox BE, Braud VM, Jakobsen BK, Stuart DI, McMichael AJ, Bell JI, Jones EY. 1998. Structural features impose tight peptide binding specificity in the nonclassical MHC molecule HLA-E. Mol Cell, 1 (4), pp. 531-541. | Show Abstract | Read more

The crystal structure of the nonclassical human class lb MHC molecule HLA-E has been determined in complex with a prototypic ligand, the nonamer peptide (VMAPRTVLL), derived from the highly conserved residues 3-11 of the human MHC class la leader sequence. The mode of peptide binding retains some of the standard features observed in MHC class la complexes, but novel features imply that HLA-E has evolved to mediate specific binding to a tightly defined set of almost identical hydrophobic peptides from the highly conserved class l leader sequences. These molecular adaptations make HLA-E a rigorous checkpoint at the cell surface reporting on the integrity of the antigen processing pathway to CD94/NKG2 receptor-bearing natural killer cells.

Crocker PR, Clark EA, Filbin M, Gordon S, Jones Y, Kehrl JH, Kelm S, Le Douarin N, Powell L, Roder J et al. 1998. Siglecs: a family of sialic-acid binding lectins. Glycobiology, 8 (2), pp. v. | Read more

Sharma A, Rao Z, Fry E, Booth T, Jones EY, Rowlands DJ, Simmons DL, Stuart DI. 1997. Specific interactions between human integrin alpha v beta 3 and chimeric hepatitis B virus core particles bearing the receptor-binding epitope of foot-and-mouth disease virus. Virology, 239 (1), pp. 150-157. | Show Abstract | Read more

Purified integrin alpha v beta 3 was used in solid-phase binding studies with chimeric hepatitis B cores which carry the RGD-containing loop of VP1 protein of the foot-and-mouth disease virus (FMDV). High levels of specific binding between the integrin and the particles were detected by enzyme-linked immunosorbent assays. The binding was Mn2+ cation dependent and could be competed with fibronectin, vitronectin, and the peptide GRGDSPK. Particles in which the RGD motif had been mutated to RGE failed to bind, indicating that the chimeric cores bound specifically to the ligand binding site of integrin alpha v beta 3. Electron micrographs showed several individual alpha v beta 3 molecules bound to the surface of each chimeric particle. Collectively, these data constitute firm evidence that the RGD-containing loop of FMDV is critical for binding to alpha v beta 3 and provide support for identification of alpha v beta 3 as a potential cellular receptor for FMDV.

Crocker PR, May A, Jones EY. 1997. Structural basis of sialoadhesin-mediated cell-cell interactions GLYCOBIOLOGY, 7 (7), pp. 4-4.

Burtnick LD, Koepf EK, Grimes J, Jones EY, Stuart DI, McLaughlin PJ, Robinson RC. 1997. The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation. Cell, 90 (4), pp. 661-670. | Show Abstract | Read more

The structure of gelsolin has been determined by crystallography and comprises six structurally related domains that, in a Ca2+-free environment, pack together to form a compact globular structure in which the putative actin-binding sequences are not sufficiently exposed to enable binding to occur. We propose that binding Ca2+ can release the connections that join the N- and C-terminal halves of gelsolin, enabling each half to bind actin relatively independently. Domain shifts are proposed in response to Ca2+ as bases for models of how gelsolin acts to sever, cap, or nucleate F-actin filaments. The structure also invites discussion of polyphosphoinositide binding to segment 2 and suggests how mutation at Asp-187 could initiate a series of events that lead to deposition of amyloid plaques, as observed in victims of familial amyloidosis (Finnish type).

Newton JP, Buckley CD, Jones EY, Simmons DL. 1997. Residues on both faces of the first immunoglobulin fold contribute to homophilic binding sites of PECAM-1/CD31. J Biol Chem, 272 (33), pp. 20555-20563. | Show Abstract | Read more

CD31 (PECAM-1) is a member of the immunoglobulin superfamily whose extracellular domain is comprised of six immunoglobulin-like domains. It is widely expressed on endothelium, platelets, around 50% of lymphocytes, and cells of myeloid lineage. CD31 has been shown to be involved in interendothelial adhesion and leukocyte-endothelial interactions, particularly during transmigration. CD31-mediated adhesion is complex, because CD31 is capable of mediating both homophilic and multiple heterophilic adhesive interactions. Here we show that the NH2-terminal (membrane-distal) immunoglobulin domain of CD31 is necessary but not sufficient to support stable homophilic adhesion. Key residues forming the binding site within this domain have been identified by analysis of 26 single point mutations, representing the most systematic analysis of a fully homophilic interaction between immunoglobulin superfamily family members to date. This revealed five mutations that affect homophilic binding. Uniquely, the residues involved are exposed on both faces of the immunoglobulin fold, leading us to propose a novel mechanism for CD31 homophilic adhesion.

Newham P, Craig SE, Seddon GN, Schofield NR, Rees A, Edwards RM, Jones EY, Humphries MJ. 1997. Alpha4 integrin binding interfaces on VCAM-1 and MAdCAM-1. Integrin binding footprints identify accessory binding sites that play a role in integrin specificity. J Biol Chem, 272 (31), pp. 19429-19440. | Show Abstract | Read more

Integrins are a family of heterodimeric adhesion receptors that mediate cellular interactions with a range of matrix components and cell surface proteins. Vascular cell adhesion molecule-1 (VCAM-1) is an endothelial cell ligand for two leukocyte integrins (alpha4beta1 and alpha4beta7). A related CAM, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is recognized by alpha4beta7 but is a poor ligand for alpha4beta1. Previous studies have revealed that all alpha4 integrin-ligand interactions are dependent on a key acidic ligand motif centered on the CAM domain 1 C-D loop region. By generating VCAM-1/MAdCAM-1 chimeras and testing recombinant proteins in cell adhesion assays we have found that alpha4beta1 binds to the MAdCAM-1 adhesion motif when present in VCAM-1, but not when the VCAM-1 motif was present in MAdCAM-1, suggesting that this region does not contain all of the information necessary to determine integrin binding specificity. To characterize integrin-CAM specificity further we measured alpha4beta1 and alpha4beta7 binding to a comprehensive set of mutant VCAM-1 constructs containing amino acid substitutions within the predicted integrin adhesion face. These data revealed the presence of key "regulatory residues" adjacent to integrin contact sites and an important difference in the "footprint" of alpha4beta1 and alpha4beta7 that was associated with an accessory binding site located in VCAM-1 Ig domain 2. The analogous region in MAdCAM-1 is markedly different in size and sequence and when mutated abolishes integrin binding activity.

Gao GF, Tormo J, Gerth UC, Wyer JR, McMichael AJ, Stuart DI, Bell JI, Jones EY, Jakobsen BK. 1997. Crystal structure of the complex between human CD8alpha(alpha) and HLA-A2. Nature, 387 (6633), pp. 630-634. | Show Abstract | Read more

The dimeric cell-surface glycoprotein CD8 is crucial to the positive selection of cytotoxic T cells in the thymus. The homodimer CD8alpha(alpha) or the heterodimer alpha beta stabilizes the interaction of the T-cell antigen receptor (TCR) with major histocompatibility complex (MHC) class I/peptide by binding to the class I molecule. Here we report the crystal structure at 2.7 A resolution of a complex between CD8alpha(alpha) and the human MHC molecule HLA-A2, which is associated with peptide. CD8alpha(alpha) binds one HLA-A2/peptide molecule, interfacing with the alpha2 and alpha3 domains of HLA-A2 and also contacting beta2-microglobulin. A flexible loop of the alpha3 domain (residues 223-229) is clamped between the complementarity-determining region (CDR)-like loops of the two CD8 subunits in the classic manner of an antibody-antigen interaction, precluding the binding of a second MHC molecule. The position of the alpha3 domain is different from that in uncomplexed HLA-A2, being most similar to that in the TCR/Tax/HLA-A2 complex, but no conformational change extends to the MHC/peptide surface presented for TCR recognition. Although these shifts in alpha3 may provide a synergistic modulation of affinity, the binding of CD8 to MHC is clearly consistent with an avidity-based contribution from CD8 to TCR-peptide-MHC interactions.

Goulder PJ, Reid SW, Price DA, O'Callaghan CA, McMichael AJ, Phillips RE, Jones EY. 1997. Combined structural and immunological refinement of HIV-1 HLA-B8-restricted cytotoxic T lymphocyte epitopes. Eur J Immunol, 27 (6), pp. 1515-1521. | Show Abstract | Read more

This study demonstrates that use of structural information improves the definition and optimization of cytotoxic T lymphocyte (CTL) epitopes. Epitope optimization usually requires numerous truncated peptides or a reverse immunogenetic approach, where the peptide binding motif is used to predict epitopes. These binding motifs do not reliably predict all peptides which are CTL epitopes. Comparison of 24 peptides eluted from HLA-B8 with 10 HLA-B8-restricted defined CTL epitopes demonstrated that known epitopes varied considerably at anchor positions. We used structural information based on determination of the crystal structure of the HLA-B8-GGKKKYKL complex to reassess previously described CTL epitopes, to predict new epitopes, and to predict the consequences of naturally occurring variation within epitopes. These predictions were confirmed by cytotoxicity and binding assays. Use of combined structural and immunological data more accurately defines the true peptide-binding motif of a restriction element than eluted peptide data allows.

Vinson M, Mucklow S, May AP, Jones EY, Kelm S, Crocker PR. 1997. Sialic acid recognition by sialoadhesin and related lectins TRENDS IN GLYCOSCIENCE AND GLYCOTECHNOLOGY, 9 (47), pp. 283-297. | Show Abstract | Read more

Sialic acids are abundant on the surface of mammalian cells where, through their negative charge, they assist in the prevention of undesirable cell-cell interactions. The recent discovery of the sialoadhesin subset of the immunoglobulin super-family, which includes sialoadhesin, CD22, myelin associated glycoprotein (MAG) and CD33, has raised the possibility that sialic acids are also instrumental in promoting cell-cell interactions in a variety of physiological systems. Each of these membrane proteins exhibits a distinct specificity for both the type of sialic acid recognised and its linkage to subterminal sugars. They share a high degree of sequence similarity within the NH 2 -terminal two Ig domains which also display a unique arrangement of conserved disulphide bonds. Domains 1 and 2 (numbering from the NH 2 -terminus) are likely to be representative of an ancestral gene that gave rise to the current members of the family through gene duplication. By generating truncated recombinant proteins, together with site-directed mutagenesis, the GFCC'C" faces of the NH 2 -terminal V-set domains of sialoadhesin and CD22 have been shown to contain the sialic acid binding site. Recently, this has been confirmed by X-ray crystallography which has led to the structural determination of the V-set domain of sialoadhesin complexed with a ligand, 3' sialyllactose. The similarities and differences in sialic acid recognition by sialoadhesin and other sialic acid binding proteins are discussed.

Braud V, Jones EY, McMichael A. 1997. The human major histocompatibility complex class Ib molecule HLA-E binds signal sequence-derived peptides with primary anchor residues at positions 2 and 9. Eur J Immunol, 27 (5), pp. 1164-1169. | Show Abstract | Read more

Human histocompatibility leukocyte antigen E (HLA-E) and mouse major histocompatibility complex (MHC) class Ib antigen, Qa-1, share the same substitutions at two normally conserved positions 143 and 147, which are likely to affect binding of the C terminus of peptides. Qa-1 is able to bind a peptide derived from the leader sequence of H-2 D and H-2 L molecules. We developed a peptide binding assay in vitro to compare the binding specificity of HLA-E with the mouse MHC class Ib molecule Qa-1. We demonstrate that HLA-E binds, although poorly, the peptide which binds to Qa-1 and that it also binds nonamer signal sequence-derived peptides from human MHC class I molecules. Using alanine and glycine substitutions, we could define primary anchor residues at positions 2 and 9 and secondary anchor residues at position 7 and possibly 3.

Esnouf RM, Ren J, Hopkins AL, Ross CK, Jones EY, Stammers DK, Stuart DI. 1997. Unique features in the structure of the complex between HIV-1 reverse transcriptase and the bis(heteroaryl)piperazine (BHAP) U-90152 explain resistance mutations for this nonnucleoside inhibitor. Proc Natl Acad Sci U S A, 94 (8), pp. 3984-3989. | Show Abstract | Read more

The viral reverse transcriptase (RT) provides an attractive target in the search for anti-HIV therapies. The nonnucleoside inhibitors (NNIs) are a diverse set of compounds (usually HIV-1 specific) that function by distorting the polymerase active site upon binding in a nearby pocket. Despite being potent and of generally low toxicity, their clinical use has been limited by rapid selection for resistant viral populations. The 2.65-A resolution structure of the complex between HIV-1 RT and the bis(heteroaryl)piperazine (BHAP) NNI, 1-(5-methanesulfonamido-1H-indol-2-yl-carbonyl)-4- [3-(1-methyl-ethylamino) pyridinyl] piperazine (U-90152), reveals the inhibitor conformation and bound water molecules. The bulky U-90152 molecule occupies the same pocket as other NNIs, but the complex is stabilized quite differently, in particular by hydrogen bonding to the main chain of Lys-103 and extensive hydrophobic contacts with Pro-236. These interactions rationalize observed resistance mutations, notably Pro-236-Leu, which occurs characteristically for BHAPs. When bound, part of U-90152 protrudes into the solvent creating a channel between Pro-236 and the polypeptide segments 225-226 and 105-106, giving the first clear evidence of the entry mode for NNIs. The structure allows prediction of binding modes for related inhibitors [(altrylamino)piperidine-BHAPs] and suggests changes to U-90152, such as the addition of a 6 amino group to the pyridine ring, which may make binding more resilient to mutations in the RT. The observation of novel hydrogen bonding to the protein main chain may provide lessons for the improvement of quite different inhibitors.

Stuart DI, Jones EY. 1997. Cutting complexity down to size. Nature, 386 (6624), pp. 437-438. | Show Abstract | Read more

The proteasome is the most complex eukaryotic macromolecular assembly yet seen in fine detail. The structure reveals completely unexpected mechanisms by which the proteasome neatly chops up unwanted proteins for disposal or display to the immune system.

May AP, Robinson RC, Aplin RT, Bradfield P, Crocker PR, Jones EY. 1997. Expression, crystallization, and preliminary X-ray analysis of a sialic acid-binding fragment of sialoadhesin in the presence and absence of ligand. Protein Sci, 6 (3), pp. 717-721. | Show Abstract | Read more

Sialoadhesin is a macrophage-restricted cell surface receptor, consisting of 17 immunoglobulin domains, which mediates cell adhesion via the recognition of specific sialylated glycoconjugates. A functional fragment of sialoadhesin, comprising the N-terminal immunoglobulin domain, has been expressed in Chinese hamster ovary cells as both native (SnD1) and selenomethionyl (Se-SnD1) stop protein. The successful production of 86% selenomethionine-incorporated protein represents a rare example of production of selenium-labeled protein in mammalian cells. SnD1 and Se-SnD1 have been crystallized in the absence of ligand, and SnD1 has also been crystallized in the presence of its ligand 2,3 sialyllactose. The ligand-free crystals of SnD1 and Se-SnD1 were isomorphous, of space group P3(1)21 or P3(2)21, with unit cell dimensions a = b 38.9 A,c = 152.6 A, alpha = beta = 90 degrees, gamma = 120 degrees, and diffracted to a maximum resolution of 2.6 A. Cocrystals containing 2,3 sialyllactose diffracted to 1.85 A at a synchrotron source and belong to space group P2(1)2(1)2(1), with unit cell dimensions a = 40.9 A, b = 97.6 A,c = 101.6 A, alpha = beta = gamma = 90 degrees.

Jones EY. 1997. MHC class I and class II structures. Curr Opin Immunol, 9 (1), pp. 75-79. | Show Abstract | Read more

The basic structures of MHC class I and class II molecules are now well established. Over the past twelve months structural data on MHC class I molecules have provided details of the peptide binding groove for a number of alleles and have elaborated the mechanisms that allow binding of a range of peptides. Recent MHC class II structures have illustrated the mode of peptide binding both in mature complexes and in the MHC class II complex with a fragment of invariant chain (CLIP) during maturation.

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Gao GF, Tormo J, Gerth UC, Wyer JR, McMichael AJ, Stuart DI, Bell JI, Jones EY, Jakobsen BK. 1997. Crystal structure of the complex between CD8αα human and HLA-A2 Nature, 387 (6633), pp. 630-634. | Show Abstract | Read more

The dimeric cell-surface glycoprotein CD8 is crucial to the positive selection of cytotoxic T cells in the thymus. The homodimer CD8αα or the heterodimer αβ stabilizes the interaction of the T-cell antigen receptor (TCR) with major histocompatibility complex (MHC) class l/peptide by binding to the class I molecule. Here we report the crystal structure at 2.7 Å resolution of a complex between CD8αα and the human MHC molecule HLA-A2, which is associated with peptide. CD8αα binds one HLA-A2/peptide molecule, interfacing with the α2 and α3 domains of HLA-A2 and also contacting β 2 - microglobulin. A flexible loop of the α3 domain (residues 223-229) is damped between the complementarity-determining region (CDR)-like loops of the two CD8 subunits in the classic manner of an antibody-antigen interaction, precluding the binding of a second MHC molecule. The position of the α3 domain is different from that in uncomplexed HLA-A2 (refs 3, 4), being most similar to that in the TCR/Tax/HLA-A2 complex, but no conformational change extends to the MHC/peptide surface presented for TCR recognition. Although these shifts in α3 may provide a synergistic modulation of affinity, the binding of CD8 to MHC is dearly consistent with an avidity-based contribution from CD8 to TCR-peptide-MHC interactions.

Chothia C, Jones EY. 1997. The molecular structure of cell adhesion molecules. Annu Rev Biochem, 66 (1), pp. 823-862. | Show Abstract | Read more

Considerable advances have been made in our knowledge of the molecular structure of cell adhesion molecules, their binding sites, and adhesion complexes. For the cadherins, protein zero, and CD2, additional experimental data support the insights obtained from structural analysis of their domains and molecular models of their adhesion complexes. For neural cell adhesion molecules, L1, fibronectin, tenascin-C, integrins, and vascular cell adhesion molecules, the molecular structure of domains, and in most cases their binding sites, have been elucidated. The substrate recognition sites in some of these molecules possess rate constants for association and dissociation that permit both rapid cell migration and, through avidity, high-affinity cell-cell interactions.

Reid SW, McAdam S, Smith KJ, Klenerman P, O'Callaghan CA, Harlos K, Jakobsen BK, McMichael AJ, Bell JI, Stuart DI, Jones EY. 1996. Antagonist HIV-1 Gag peptides induce structural changes in HLA B8. J Exp Med, 184 (6), pp. 2279-2286. | Show Abstract | Read more

In the cellular immune response, recognition by CTL-TCRs of viral antigens presented as peptides by HLA class I molecules, triggers destruction of the virally infected cell (Townsend, A.R.M., J. Rothbard, F.M. Gotch, G. Bahadur, D. Wraith, and A.J. McMichael. 1986. Cell. 44:959-968). Altered peptide ligands (APLs) which antagonise CTL recognition of infected cells have been reported (Jameson, S.C., F.R. Carbone, and M.J. Bevan. 1993. J. Exp. Med. 177:1541-1550). In one example, lysis of antigen presenting cells by CTLs in response to recognition of an HLA B8-restricted HIV-1 P17 (aa 24-31) epitope can be inhibited by naturally occurring variants of this peptide, which act as TCR antagonists (Klenerman, P., S. Rowland Jones, S. McAdam, J. Edwards, S. Daenke, D. Lalloo, B. Koppe, W. Rosenberg, D. Boyd, A. Edwards, P. Giangrande, R.E. Phillips, and A. McMichael. 1994. Nature (Lond.). 369:403-407). We have characterised two CTL clones and a CTL line whose interactions with these variants of P17 (aa 24-31) exhibit a variety of responses. We have examined the high resolution crystal structures of four of these APLs in complex with HLA B8 to determine alterations in the shape, chemistry, and local flexibility of the TCR binding surface. The variant peptides cause changes in the recognition surface by three mechanisms: changes contributed directly by the peptide, effects transmitted to the exposed peptide surface, and induced effects on the exposed framework of the peptide binding groove. While the first two mechanisms frequently lead to antagonism, the third has more profound effects on TCR recognition.

Jones EY. 1996. Three-dimensional structure of cell adhesion molecules. Curr Opin Cell Biol, 8 (5), pp. 602-608. | Show Abstract | Read more

Recent structural data have provided insights into various forms of specific cell adhesion interactions, including both protein-protein and protein-glycoconjugate recognition events. The major advances have been made in the structural characterization of cadherin-cadherin and integrin-ligand mediated adhesion.

Robinson RC, Radziejewski C, Stuart DI, Jones EY. 1996. Crystals of the neurotrophins. Protein Sci, 5 (5), pp. 973-977. | Show Abstract | Read more

The neurotrophins show a high degree of amino acid sequence homology, share similar solution properties, and display distinct but parallel functionalities. Here we report the crystallization and preliminary X-ray characterization of three neurotrophins: brain-derived neurotrophin, neurotrophin 3, and the heterodimer between brain-derived neurotrophin and neurotrophin 4. These findings are related to other published crystal parameters for neurotrophins, leading to the observation that, although crystal packing is highly variant, neurotrophins share common solubilities with respect to crystal growth.

Jones EY. 1996. Structural biology of IgSF cell adhesion molecules. FASEB JOURNAL, 10 (6), pp. 2135-2135.

Vinson M, van der Merwe PA, Kelm S, May A, Jones EY, Crocker PR. 1996. Characterization of the sialic acid-binding site in sialoadhesin by site-directed mutagenesis. J Biol Chem, 271 (16), pp. 9267-9272. | Show Abstract | Read more

The sialoadhesins are a distinct subgroup of the immunoglobulin superfamily, comprising sialoadhesin, CD22, the myelin-associated glycoprotein, and CD33. They can all mediate sialic acid-dependent binding to cells with distinct specificities. Sialoadhesin is a murine macrophage-restricted cell-surface molecule with 17 extracellular immunoglobulin-like domains that recognizes NeuAc alpha 2-3Gal in N- and O-glycans and interacts preferentially with cells of the granulocytic lineage. Its sialic acid-binding site is located within the NH2-terminal (membrane-distal) V-set domain. Here we have carried out site-directed mutagenesis in an attempt to identify the binding site of sialoadhesin. A subset of nonconservative mutations disrupted sialic acid-dependent binding without affecting binding of three monoclonal antibodies directed to two distinct epitopes of sialoadhesin. A CD8 alpha-based molecular model predicts that these residues form a contiguous binding site on the GFCC'C" beta-sheet of the V-set domain centered around an arginine in the F strand. A conservative mutation of this arginine to lysine also abolished binding. This amino acid is conserved among all members of the sialoadhesin family and is therefore likely to be a key residue in mediating sialic acid-dependent binding of sialoadhesins to cells.

Hopkins AL, Ren J, Esnouf RM, Willcox BE, Jones EY, Ross C, Miyasaka T, Walker RT, Tanaka H, Stammers DK, Stuart DI. 1996. Complexes of HIV-1 reverse transcriptase with inhibitors of the HEPT series reveal conformational changes relevant to the design of potent non-nucleoside inhibitors. J Med Chem, 39 (8), pp. 1589-1600. | Show Abstract | Read more

Crystal structures of HIV-1 reverse transcriptase (RT) complexed with a range of chemically diverse non-nucleoside inhibitors (NNIs) have shown a single pocket in which the inhibitors bind and details of the inhibitor-protein interactions. To delineate the structural requirements for an effective inhibitor, we have determined the structures of three closely related NNIs which vary widely in their potencies. Crystal structures of HIV-1 RT complexed with two very potent inhibitors, MKC-442 and TNK-651, at 2.55 angstroms resolution complement our previous analysis of the complex with the less effective inhibitor, HEPT. These structures reveal conformational changes which correlate with changes in potency. We suggest that a major determinant of increased potency in the analogues of HEPT is an improved interaction between residue Tyr181 in the protein and the 6-benzyl ring of the inhibitors which stabilizes the structure of the complex. This arises through a conformational switching of the protein structure triggered by the steric bulk of the 5-substituent of the inhibitor pyrimidine ring.

Reid SW, Smith KJ, Jakobsen BK, O'Callaghan CA, Reyburn H, Harlos K, Stuart DI, McMichael AJ, Bell JI, Jones EY. 1996. Production and crystallization of MHC class I B allele single peptide complexes. FEBS Lett, 383 (1-2), pp. 119-123. | Show Abstract | Read more

Major histocompatibility complex class I B alleles, HLA B8, B53 and B3501 have been cloned, expressed, refolded and crystallized in specific complexes with a number of different 8-mer and 9-mer peptides. For some of these crystallization was initiated by cross-seeding between different B allele complexes. All crystallize in the space group P212121, with similar unit cell dimensions of approximately 52 A X 81 A X 112 A, contain one complex per asymmetric unit and diffract to approximately 2.0 A resolution.

Smith KJ, Reid SW, Stuart DI, McMichael AJ, Jones EY, Bell JI. 1996. An altered position of the alpha 2 helix of MHC class I is revealed by the crystal structure of HLA-B*3501. Immunity, 4 (3), pp. 203-213. | Show Abstract | Read more

The crystal structure of the human major histocompatibility complex class I B allele HLA B*3501 complexed with the 8-mer peptide epitope HIV1 Nef 75-82 (VPLRPMTY) has been determined at 2.0 angstrom resolution. Comparison with the crystal structure of the closely related allele HLA B*5301 reveals the structural basis for the tyrosine specificity of the B*3501 F pocket. The structure also reveals a novel conformation of the 8-mer peptide within the binding groove. The positions of the peptide N and C termini are nonstandard, but the classic pattern of hydrogen bonding to nonpolymorphic MHC class I residues is maintained, at the N terminus by addition of a water molecule, and at the C terminus by a substantial shift in the alpha 2 helix.

Smith KJ, Reid SW, Harlos K, McMichael AJ, Stuart DI, Bell JI, Jones EY. 1996. Bound water structure and polymorphic amino acids act together to allow the binding of different peptides to MHC class I HLA-B53. Immunity, 4 (3), pp. 215-228. | Show Abstract | Read more

The structure of the human MHC class I molecule HLA-B53 complexed to two nonameric peptide epitopes (from the malaria parasite P. falciparum and the HIV2 gag protein) has been determined by X-ray crystallography at 2.3 angstrom resolution. The structures reveal the architecture of a Pro-specific B pocket common to many HLA-B alleles. Relative to other alleles, the B53 peptide-binding groove is widened by a significant (up to 1.25 angstrom) shift in the position of the alpha 1 helix. Within this groove, bound water molecules, acting in concert with the side chains of polymorphic residues, provide the functional malleability of the MHC, which enables the high affinity/low specificity binding of multiple peptide epitopes.

Stuart DI, Jones EY. 1995. Recognition at the cell surface: recent structural insights. Curr Opin Struct Biol, 5 (6), pp. 735-743. | Show Abstract | Read more

In the past few years, structural biology has begun to reveal details of some of the receptors and associated interactions responsible for protein-mediated recognition events at the cell surface. Recent data span the fields of cytokine-receptor interactions, cell adhesion molecules and viral infection.

Ren J, Esnouf R, Hopkins A, Ross C, Jones Y, Stammers D, Stuart D. 1995. The structure of HIV-1 reverse transcriptase complexed with 9-chloro-TIBO: lessons for inhibitor design. Structure, 3 (9), pp. 915-926. | Show Abstract | Read more

BACKGROUND: HIV reverse transcriptase (RT) is a key target of anti-AIDS therapies. Structural studies of HIV-1 RT, unliganded and complexed with different non-nucleoside inhibitors (NNIs), have pointed to a common mode of binding and inactivation through distortion of the polymerase catalytic site by NNIs containing two hinged rings. The mode of binding of the TIBO family of inhibitors is of interest because these compounds do not fit the two-hinged-ring model. RESULTS: The structure of HIV-1 RT complexed with 9-chloro-TIBO (R82913) has been determined at 2.6 A resolution. As reported for the lower resolution analysis of another TIBO compound, this inhibitor binds at the same site as other NNIs, but our higher resolution study reveals the Cl-TIBO is distorted from the conformation seen in crystals of the inhibitor alone. This allows Cl-TIBO to mimic the binding of NNIs containing two hinged rings. Inhibitor-protein interactions are again predominantly hydrophobic and the protein conformation corresponds to that seen in complexes with other tight-binding NNIs. CONCLUSIONS: Although Cl-TIBO is chemically very different from other NNIs, it achieves remarkable spatial equivalence and shape complementarity with other NNIs on binding to RT. Comparison of the different RT-NNI complexes suggests modifications to the TIBO group of inhibitors which might enhance their binding and hence, potentially, their therapeutic efficacy.

Robinson RC, Grey LM, Staunton D, Stuart DI, Heath JK, Jones EY. 1995. The crystal structure of murine leukemia inhibitory factor. Ann N Y Acad Sci, 762 pp. 179-187. | Show Abstract

We have determined the structure of murine leukemia inhibitory factor (LIF) by X-ray crystallography at 2.0 A resolution. The current crystal structure comprises native LIF residues 9 to 180 with 40 ordered water molecules. For this model the R value (with a bulk solvent correction) is 18.6% on all data from 20.0 A to 2.0 A with stereochemistry typified by root mean square deviations from ideal bond lengths of 0.015 A. The mainchain fold conforms to the four alpha-helix bundle topology previously observed for several members of the hematopoietic cytokine family. Of these, LIF shows closest structural homology to granulocyte colony stimulating factor and growth hormone. Sequence alignments for the functionally related molecules oncostatin M and ciliary neurotrophic factor, when mapped to the LIF structure, indicate regions of conserved structural and surface character. Analysis of published mutagenesis data implicate two regions of receptor interaction which are located in the fourth helix and the preceding loop. A model for receptor binding based on the structure of the growth hormone ligand/receptor complex requires additional, novel features to account for these data.

Spraggon G, Phillips C, Nowak UK, Ponting CP, Saunders D, Dobson CM, Stuart DI, Jones EY. 1995. The crystal structure of the catalytic domain of human urokinase-type plasminogen activator. Structure, 3 (7), pp. 681-691. | Show Abstract | Read more

BACKGROUND: Urokinase-type plasminogen activator (u-PA) promotes fibrinolysis by catalyzing the conversion of plasminogen to the active protease plasmin via the cleavage of a peptide bond. When localized to the external cell surface it contributes to tissue remodelling and cellular migration; inhibition of its activity impedes the spread of cancer. u-PA has three domains: an N-terminal receptor-binding growth factor domain, a central kringle domain and a C-terminal catalytic protease domain. The biological roles of the fibrinolytic enzymes render them therapeutic targets, however, until now no structure of the protease domain has been available. Solution of the structure of the u-PA serine protease was undertaken to provide such data. RESULTS: The crystal structure of the catalytic domain of recombinant, non-glycosylated human u-PA, complexed with the inhibitor Glu-Gly-Arg chloromethyl ketone (EGRcmk), has been determined at a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 22.4% on all data (20.4% on data > 3 sigma). The enzyme has the expected topology of a trypsin-like serine protease. CONCLUSIONS: The enzyme has an S1 specificity pocket similar to that of trypsin, a restricted, less accessible, hydrophobic S2 pocket and a solvent-accessible S3 pocket which is capable of accommodating a wide range of residues. The EGRcmk inhibitor binds covalently at the active site to form a tetrahedral hemiketal structure. Although the overall structure is similar to that of homologous serine proteases, at six positions insertions of extra residues in loop regions create unique surface areas. One of these loop regions is highly mobile despite being anchored by the disulphide bridge which is characteristic of a small subset of serine proteases namely tissuetype plasminogen activator, Factor XII and Complement Factor I.

Wilkie AO, Morriss-Kay GM, Jones EY, Heath JK. 1995. Functions of fibroblast growth factors and their receptors. Curr Biol, 5 (5), pp. 500-507. | Show Abstract | Read more

Fibroblast growth factors were first characterized twenty years ago as mitogens of cultured fibroblasts. Despite a wealth of data from experiments in vitro, insights have begun to emerge only recently on the normal function of these growth factors in mice and humans, as a result of studies of natural and experimental mutations in the factors and their receptors.

Robinson RC, Radziejewski C, Stuart DI, Jones EY. 1995. Structure of the brain-derived neurotrophic factor/neurotrophin 3 heterodimer. Biochemistry, 34 (13), pp. 4139-4146. | Show Abstract | Read more

The development and sustenance of specific neuronal populations in the peripheral and central nervous systems are controlled through the binding of neurotrophic factors to high-affinity cell surface receptors. The neurotrophins (nerve growth factor, NGF; brain-derived neurotrophic factor, BDNF; neurotrophin 3, NT3; and neurotrophin 4, NT4) are dimeric molecules which share approximately 50% sequence identity. The crystal structure of the murine NGF homodimer [McDonald et al. (1991) Nature 354, 411-414] indicated that the dimer interface corresponds to regions of high sequence conservation throughout the neurotrophin family. This potential compatibility was duly exploited for the production in vitro of noncovalent heterodimers between the different neurotrophins [Radziejewski, C., & Robinson, R.C. (1993) Biochemistry 32, 13350-13356; Jungbluth et al. (1994) Eur. J. Biochem. 221, 677-685]. Here, we report the X-ray structure at 2.3 A resolution of one such heterodimer, between human BDNF, and human NT3. The NGF, BDNF, and NT3 protomers share the same topology and are structurally equivalent in regions which contribute to the dimer interface in line with the propensity of the neurotrophins to form heterodimers. Analysis of the structure of regions of the BDNF/NT3 heterodimer involved in receptor specificity led us to conclude that heterodimer binding to p75 involves distant binding sites separately located on each protomer of the heterodimer. In contrast, heterodimer interactions with the trk receptors probably utilize hybrid binding sites comprised of residues contributed by both protomers in the heterodimer. The existence of such hybrid binding sites for the trk receptor provides an explanation for the lower activity of the BDNF/NT3 heterodimer in comparison to the homodimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Esnouf R, Ren J, Ross C, Jones Y, Stammers D, Stuart D. 1995. Mechanism of inhibition of HIV-1 reverse transcriptase by non-nucleoside inhibitors. Nat Struct Biol, 2 (4), pp. 303-308. | Show Abstract | Read more

The structure of unliganded HIV-1 reverse transcriptase has been determined at 2.35 A resolution and refined to an R-factor of 0.219 (for all data) with good stereochemistry. The unliganded structure was produced by soaking out a weak binding non-nucleoside inhibitor, HEPT, from pregrown crystals. Comparison with the structures of four different RT and non-nucleoside inhibitor complexes reveals that only minor domain rearrangements occur, but there is a significant repositioning of a three-stranded beta-sheet in the p66 subunit (containing the catalytic aspartic acid residues 110, 185 and 186) with respect to the rest of the polymerase site. This suggests that NNIs inhibit RT by locking the polymerase active site in an inactive conformation, reminiscent of the conformation observed in the inactive p51 subunit.

Ren J, Esnouf R, Garman E, Somers D, Ross C, Kirby I, Keeling J, Darby G, Jones Y, Stuart D. 1995. High resolution structures of HIV-1 RT from four RT-inhibitor complexes. Nat Struct Biol, 2 (4), pp. 293-302. | Show Abstract | Read more

We have determined the structures of four complexes of HIV-1 reverse transcriptase with non-nucleoside inhibitors, three fully refined at high resolution. The highest resolution structure is of the RT-nevirapine complex which has an R-factor of 0.186 and a root-mean-square bond length deviation of 0.015 A for all data to 2.2 A. The structures reveal a common mode of binding for these chemically diverse compounds. The common features of binding are largely hydrophobic interactions and arise from induced shape complementarity achieved by conformational rearrangement of the enzyme and conformational/configurational rearrangement of the compounds.

Jones EY, Harlos K, Bottomley MJ, Robinson RC, Driscoll PC, Edwards RM, Clements JM, Dudgeon TJ, Stuart DI. 1995. Crystal structure of an integrin-binding fragment of vascular cell adhesion molecule-1 at 1.8 A resolution. Nature, 373 (6514), pp. 539-544. | Show Abstract | Read more

The cell-surface glycoprotein vascular cell adhesion molecule-1 (VCAM-1; ref. 1) mediates intercellular adhesion by specific binding to the integrin very-late antigen-4 (VLA-4, alpha 4 beta 1; ref. 3). VCAM-1, with the intercellular adhesion molecules ICAM-1, ICAM-2, ICAM-3 and the mucosal vascular addressin MAd-CAM-1, forms an integrin-binding subgroup of the immunoglobulin superfamily. In addition to their clinical relevance in inflammation, these molecules act as cellular receptors for viral and parasitic agents. The predominant form of VCAM-1 in vivo has an amino-terminal extracellular region comprising seven immunoglobulin-like domains. Functional studies have identified a conserved integrin-binding motif in domains 1 and 4, variants of which are present in the N-terminal domain of all members of the immunoglobulin superfamily subgroup. We report here the crystal structure of a VLA-4-binding fragment composed of the first two domains of VCAM-1. The integrin-binding motif (Q38IDSPL) is highly exposed and forms the N-terminal region of the loop between beta-strands C and D of domain 1. This motif exhibits a distinctive conformation which we predict will be common to all the integrin-binding IgSF molecules. These, and additional data, map VLA-4 binding to the face of the CFG beta-sheet, the surface previously identified as the site for intercellular adhesive interactions between members of the immunoglobulin superfamily.

Davis SJ, Davies EA, Barclay AN, Daenke S, Bodian DL, Jones EY, Stuart DI, Butters TD, Dwek RA, van der Merwe PA. 1995. Ligand binding by the immunoglobulin superfamily recognition molecule CD2 is glycosylation-independent. J Biol Chem, 270 (1), pp. 369-375. | Show Abstract | Read more

The evolutionary success of the immunoglobulin superfamily (IgSF) is thought to reflect the ability of IgSF protein domains to form stable structural units. The role of glycosylation in stabilizing these domains is controversial, however. In this study a systematic analysis of the effect of glycosylation on the ligand-binding properties of the cell-cell recognition molecule CD2, which consists of two IgSF domains, was undertaken. A form of human soluble CD2 (hsCD2) with single N-acetylglucosamine residues at each glycosylation site was produced by inhibiting glucosidase I with N-butyldeoxynojirimycin during expression in Chinese hamster ovary cells and digesting the expressed hsCD2 with endoglycosidase H. The ligand and antibody binding properties of this form of hsCD2 were indistinguishable from those of fully glycosylated hsCD2 as determined by surface plasmon resonance analyses. The protein also formed diffraction quality crystals and analysis of the 2.5-A resolution crystal structure indicated that the single N-acetylglucosamine residue present on domain 1 is unlikely to stabilize the ligand binding face of hsCD2. A second, fully deglycosylated form of hsCD2 also bound the ligand and antibodies although this form of the protein tended to aggregate. In contrast to the results of previous studies, the current data indicate that the structural integrity and ligand binding function of human CD2 are glycosylation-independent.

Bottomley MJ, Robinson RC, Driscoll PC, Harlos K, Stuart DI, Aplin RT, Clements JM, Jones EY, Dudgeon TJ. 1994. Crystallization and preliminary X-ray diffraction characterisation of both a native and selenomethionyl VLA-4 binding fragment of VCAM-1. J Mol Biol, 244 (4), pp. 464-468. | Show Abstract | Read more

Soluble fragments of the extracellular region of vascular cell adhesion molecule 1 (VCAM-1) expressed in Escherichia coli retain functional adhesive activity. An integrin (VLA-4) binding fragment consisting of the N-terminal two immunoglobulin-like domains (VCAM-d1,2) has been crystallized. The crystals belong to space group P2(1)2(1)2(1) with cell dimensions of a = 52.7 A, b = 66.5 A, c = 113.2 A and contain two molecules in the crystallographic asymmetric unit. A batch of protein produced in the standard E. coli strain (HW1110), but grown in the presence of selenomethionine enriched media, showed 85% incorporation of selenium in place of sulphur at methionine residues. The selenomethionyl VCAM-d1,2 was crystallized by microseeding techniques initially using the native crystals for nucleation. Both native and selenomethionyl crystals diffract X-rays to a minimum Bragg spacing of 1.8 A.

Stammers DK, Somers DO, Ross CK, Kirby I, Ray PH, Wilson JE, Norman M, Ren JS, Esnouf RM, Garman EF. 1994. Crystals of HIV-1 reverse transcriptase diffracting to 2.2 A resolution. J Mol Biol, 242 (4), pp. 586-588. | Show Abstract | Read more

Reverse transcriptase (RT) from the human immunodeficiency virus type 1 has been crystallized in four closely related forms, the best of which diffract X-rays to 2.2 A resolution. The RT was crystallized as a complex with a non-nucleoside inhibitor, either nevirapine or a nevirapine analogue. Crystals grew from 6% PEG 3400 buffered at pH 5. These were of space group P2(1)2(1)2(1) with unit cell parameters a = 147 A, b = 112 A, c = 79 A (form A), with one RT heterodimer in the asymmetric unit. Changes in unit cell parameters and degree of crystalline order were observed on soaking pregrown crystals in various solutions, giving three further sets of unit cells. These were a = 143 A, b = 112, A, c = 79 A (form B), a = 141 A, b = 111 A, c = 73 A (form C), a = 143 A, b = 117 A, c = 66.5 A (form D). The last two forms diffract X-rays to 2.2 A resolution. Structure determinations of these latter crystal forms of RT should give a detailed atomic model for this therapeutically important drug target.

Harlos K, Martin DM, O'Brien DP, Jones EY, Stuart DI, Polikarpov I, Miller A, Tuddenham EG, Boys CW. 1994. Crystal structure of the extracellular region of human tissue factor. Nature, 370 (6491), pp. 662-666. | Show Abstract | Read more

Tissue factor is a cell-surface glycoprotein receptor which initiates the blood coagulation cascade after vessel injury by interacting with blood clotting factor VII/VIIa and which is implicated in various pathological processes. When bound to tissue factor, factor VII is readily converted to the active protease factor VIIa by trace amounts of factors Xa, IXa or VIIa. Human tissue factor consists of 263 residues, the first 219 of which comprise the extracellular region. We have determined the crystal structure of the extracellular region at a resolution of 2.2 A. Tissue factor consists of two immunoglobulin-like domains associated through an extensive, novel, interdomain interface region. The binding site for factor VII lies at the interface region and involves residues from domain 1 and an extended loop (binding 'finger') of domain 2. This is the first reported structure of a representative of the class 2 cytokine receptor family, which also includes interferon-alpha, interferon-gamma (refs 2, 3) and interleukin-10 (ref. 4) receptors.

Bodian DL, Jones EY, Harlos K, Stuart DI, Davis SJ. 1994. Crystal structure of the extracellular region of the human cell adhesion molecule CD2 at 2.5 A resolution. Structure, 2 (8), pp. 755-766. | Show Abstract | Read more

BACKGROUND: The T-lymphocyte antigen CD2 is an adhesion molecule implicated in immune responses in vivo. The extracellular regions of the human and rat homologues of CD2 share only 45% sequence identity and bind different protein ligands. Comparison of the human and rat soluble CD2 (sCD2) structures should provide insights into the structural basis of cell surface recognition. RESULTS: We therefore determined the crystal structure of a form of human sCD2 with single N-acetylglucosamine residues at each glycosylation site to 2.5 A resolution with an R-factor of 19.3%. It is composed of two immunoglobulin superfamily domains similar to those of rat sCD2, but the relative orientation of the domains in the two homologues differs by up to 20 degrees. An interaction involving the flat, highly charged, ligand binding GFCC'C" faces of crystallographically related human sCD2 molecules duplicates, in a different lattice, that observed in the rat sCD2 crystals. CONCLUSIONS: Intramolecular flexibility appears to be a conserved feature of CD2. The head-to-head interaction between molecules represents a general model for interactions between adhesion molecules of this structural class. Ligand specificity may be influenced by the distribution of charged residues on the binding face.

Robinson RC, Grey LM, Staunton D, Vankelecom H, Vernallis AB, Moreau JF, Stuart DI, Heath JK, Jones EY. 1994. The crystal structure and biological function of leukemia inhibitory factor: implications for receptor binding. Cell, 77 (7), pp. 1101-1116. | Show Abstract | Read more

The structure of murine leukemia inhibitory factor (LIF) has been determined by X-ray crystallography at 2.0 A resolution. The main chain fold conforms to the four alpha-helix bundle topology previously observed for several members of the hematopoietic cytokine family. Of these, LIF shows closest structural homology to granulocyte colony-stimulating factor and growth hormone (GH). Sequence alignments for the functionally related molecules oncostatin M and ciliary neurotrophic factor, when mapped to the LIF structure, indicate regions of conserved surface character. Analysis of the biological function and receptor specificity of a series of human-mouse LIF chimeras implicate two regions of receptor interaction that are located in the fourth helix and the preceding loop. A model for receptor binding based on the structure of the GH ligand-receptor complex requires additional, novel features to account for these data.

Tavernor AS, Kydd JH, Bodian DL, Jones EY, Stuart DI, Davis SJ, Butcher GW. 1994. Expression cloning of an equine T-lymphocyte glycoprotein CD2 cDNA. Structure-based analysis of conserved sequence elements. Eur J Biochem, 219 (3), pp. 969-976. | Show Abstract | Read more

An equine CD2 cDNA has been isolated by monoclonal antibody screening of a T-lymphocyte cDNA library. The cDNA contained an open reading frame of 1041 bp encoding a translated product of 347 amino acids. Northern blotting analysis revealed a single mRNA species expressed in spleen, thymus and activated peripheral lymphocytes. The predicted amino acid sequence has 50-65% identity with the human, rat and mouse CD2 sequences with greatest similarity shared with the human homologue. Evolutionarily conserved structural and functional domains in CD2 were identified by comparing the sequences of the equine, human, mouse and rat CD2 homologues in the context of the recently derived crystal structure of rat soluble CD2 [Jones, E. Y., Davis, S. J., Williams, A. F., Harlos, K. & Stuart, D. I. (1992) Nature 360, 232-239]. The key conserved features of the extracellular region included core residues necessary to preserve the structural integrity of the molecule, residues in the linker region likely to maintain the unique domain organization of CD2, an array of highly charged residues in the putative ligand-binding face of the molecule and glycosylation-signal distributions that render the putative ligand-binding GFCC'C" face of domain 1 relatively unhindered by glycosylation.

Acharya KR, Passalacqua EF, Jones EY, Harlos K, Stuart DI, Brehm RD, Tranter HS. 1994. Structural basis of superantigen action inferred from crystal structure of toxic-shock syndrome toxin-1. Nature, 367 (6458), pp. 94-97. | Show Abstract | Read more

Superantigens stimulate T cells bearing particular T-cell receptor V beta sequences, so they are extremely potent polyclonal T-cell mitogens. T-cell activation is preceded by binding of superantigens to class II major histocompatibility complex (MHC) molecules. To further the structural characterization of these interactions, the crystal structure of a toxin associated with toxic-shock syndrome, TSST-1, which is a microbial superantigen, has been determined at 2.5 A resolution. The N- and C-terminal domains of the structure both contain regions involved in MHC class II association; the C-terminal domain is also implicated in binding the T-cell receptor. Despite low sequence conservation, the TSST-1 topology is similar to the structure reported for the superantigen staphylococcal enterotoxin B4. But TSST-1 lacks several of the structural features highlighted as central to superantigen activity in the staphylococcal enterotoxin B and we therefore reappraise the structural basis of superantigen action.

Spraggon G, Stuart D, Ponting C, Finnis C, Sleep D, Jones Y. 1993. Crystallization and X-ray diffraction study of recombinant platelet-derived endothelial cell growth factor. J Mol Biol, 234 (3), pp. 879-880. | Show Abstract | Read more

Crystals of recombinant platelet-derived endothelial cell growth factor (PD-ECGF) were obtained by the hanging drop vapour diffusion technique. The crystals belong to the space group P2(1)2(1)2(1) with unit cell dimensions a = 63.7 A, b = 70.4 A, c = 219.6, alpha = beta = gamma = 90 degrees, and probably contain a single dimer in the asymmetric unit. Diffraction to a minimum Bragg spacing of 3.5 A has been obtained using a synchrotron X-ray source.

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JONES E. 1993. THE IMMUNOGLOBULIN SUPERFAMILY CURRENT OPINION IN STRUCTURAL BIOLOGY, 3 (6), pp. 846-852. | Show Abstract | Read more

The steady flow of fresh data on antibody structure and interactions has continued. Recent results have also extended the scope of the available structural information to cover other members of the immunoglobulin superfamily. This review highlights the new perspectives on the structure and functions of the immunoglobulin fold which have emerged from the latest studies. © 1993.

STUART D, JONES E. 1993. WEISSENBERG DATA-COLLECTION FOR MACROMOLECULAR CRYSTALLOGRAPHY CURRENT OPINION IN STRUCTURAL BIOLOGY, 3 (5), pp. 737-740. | Show Abstract | Read more

The Weissenberg method has recently emerged as a viable technique in protein crystallography with the demonstration of fast, efficient X-ray diffraction data collection from a variety of protein crystals by this method at the Photon Factory (Tskuba, Japan). This review assesses the technique in the context of current results and future developments. © 1993.

PICKFORD M, GARMAN E, JONES E, STUART D. 1993. A DESIGN OF CRYSTAL MOUNTING CELL THAT ALLOWS THE CONTROLLED VARIATION OF HUMIDITY AT THE PROTEIN CRYSTAL DURING X-RAY-DIFFRACTION JOURNAL OF APPLIED CRYSTALLOGRAPHY, 26 (3), pp. 465-466. | Read more

Stammers DK, Delves C, Ballantine S, Jones EY, Stuart DI, Achari A, Bryant PK, Champness JN. 1993. Preliminary crystallographic data for Pneumocystis carinii dihydrofolate reductase. J Mol Biol, 230 (2), pp. 679-680. | Show Abstract | Read more

Dihydrofolate reductase from Pneumocystis carinii has been crystallized in a form suitable for high resolution X-ray diffraction studies. Recombinant enzyme that had been refolded following solubilization in guanidinium hydrochloride was crystallized as both a ternary complex with the cofactor NADPH and the inhibitor trimethoprim as well as a binary complex with NADPH. The two types of complex crystallized isomorphously from polyethylene glycol using sitting-drop vapour diffusion. The crystals were of space group P2(1) with unit cell parameters, a = 69.9 A, b = 43.6 A, c = 37.6 A, beta = 117.7 degrees, with one molecule per asymmetric unit. The crystals diffracted to 1.8 A resolution.

Davis SJ, Puklavec MJ, Ashford DA, Harlos K, Jones EY, Stuart DI, Williams AF. 1993. Expression of soluble recombinant glycoproteins with predefined glycosylation: application to the crystallization of the T-cell glycoprotein CD2. Protein Eng, 6 (2), pp. 229-232. | Read more

JONES E, STUART D, GARMAN E, GRIEST R, PHILLIPS D, TAYLOR G, MATSUMOTO O, DARBY G, LARDER B, LOWE D et al. 1993. THE GROWTH AND CHARACTERIZATION OF CRYSTALS OF HUMAN-IMMUNODEFICIENCY-VIRUS (HIV) REVERSE-TRANSCRIPTASE JOURNAL OF CRYSTAL GROWTH, 126 (2-3), pp. 261-269. | Show Abstract | Read more

Extensive studies on the crystallization of HIV-1 reverse transcriptase (RT) have yielded several crystal forms, two of which show diffraction to minimum Bragg spacings of 6 Å or better. Type 1 crystals belong to the space group P2 1 2 1 2 1 with unit cell dimensions a = 147 A ̊, b = 190 A ̊ and c = 182 A ̊. Crystal density measurement indicate a very high crystal solvent content of 77% consistent with the presence of two RT heterodimers (66k/51k) per crystallographic asymmetric unit. These crystals are suitable for a low resolution determination of the apoenzyme structure. The second well ordered crystal form (space group P4 2 22 with unit cell dimensions a = b = 120 A ̊, c = 320 A ̊) results from the co-crystallization of RT heterodimer and a double-stranded DNA oligonucleotide. Crystal density measurements again yield a relatively high value for the solvent content (7%; one RT heterodimer per crystallographic asymmetric unit) and elemental analysis indicates that one DNA oligonucleotide is associated with each RT heterodimer. This is consistent with each heterodimer possessing a single, competent, active site. © 1993.

Jones EY, Davis SJ, Williams AF, Harlos K, Stuart DI. 1992. Crystal structure at 2.8 A resolution of a soluble form of the cell adhesion molecule CD2. Nature, 360 (6401), pp. 232-239. | Show Abstract | Read more

The crystal structure of a soluble form of the T lymphocyte antigen CD2 provides the first complete view of the extracellular region of a cell adhesion molecule. The topology of the molecule, which comprises two immunoglobulin-like domains, is the same as that of the first two domains of CD4 but the relative domain orientation is altered by a fairly flexible linker region. The putative ligand-binding beta-sheet forms a flat surface towards the top of the molecule. Crystal contacts between these surfaces suggest a plausible model for the adhesive interaction.

Jones EY, Stuart DI, Walker NP. 1992. Crystal structure of TNF. Immunol Ser, 56 pp. 93-127.

Jones EY, Walker NP, Stuart DI. 1991. Methodology employed for the structure determination of tumour necrosis factor, a case of high non-crystallographic symmetry. Acta Crystallogr A, 47 ( Pt 6) (6), pp. 753-770. | Show Abstract | Read more

The structure of the protein tumour necrosis factor (TNF) was determined from crystals of space group P3(1)21 which contain six copies of the TNF monomer per crystallographic asymmetric unit [Jones, Stuart & Walker (1989). Nature (London), 338, 225-228]. The nature of these crystals (relatively high crystallographic symmetry coupled with multiple copies of the protein in the asymmetric unit) led to some peculiarly challenging problems at several points in the structure determination. In particular, (1) self-rotation function calculations failed to yield clearly interpretable solutions, (2) the analysis of difference Patterson maps for heavy-atom derivatives required the development of a Patterson search program suite GROPAT. The redundancy in the asymmetric unit allowed refinement of poor-quality isomorphous phases at 4 A resolution and phase extension from 4 to 2.9 A resolution using real-space symmetry averaging and solvent flattening in the absence of any isomorphous phase information. Despite further difficulties caused by structural differences between the six independent copies of the monomer the resultant electron density map was of high quality and proved to be easily interpretable.

Jones EY, Miller A. 1991. Analysis of structural design features in collagen. J Mol Biol, 218 (1), pp. 209-219. | Show Abstract | Read more

The principles governing the molecular design of collagen have been investigated using molecular graphics to link the available sequence and X-ray diffraction data. The primary structure is classified into three regions of relevance to the intermolecular packing. Functionally important variations are revealed in the distribution of the amino acid residues. Solvent accessibility studies show that the formation of the triple helix is comparable to the stage of secondary structure formation in globular proteins. The remaining hydrophobic interaction potential serves to determine the three-dimensional packing of the molecules within a fibril. A model for the fibril compatible with the X-ray data is suggested. Particular attention is directed onto the boundaries of the overlap region designated the T zones. Azimuthal orientations of the helical portions at these levels appear to be locked by means of a network of aromatic interactions.

Jones EY, Walker NP, Stuart DI. 1991. Methodology employed for the structure determination of tumour necrosis factor, a case of high non-crystallographic symmetry. Acta Crystallographica Section A: Foundations of Crystallography, 47 | Show Abstract | Read more

The structure of the protein tumour necrosis factor (TNF) was determined from crystals of space group P3(1)21 which contain six copies of the TNF monomer per crystallographic asymmetric unit [Jones, Stuart & Walker (1989). Nature (London), 338, 225-228]. The nature of these crystals (relatively high crystallographic symmetry coupled with multiple copies of the protein in the asymmetric unit) led to some peculiarly challenging problems at several points in the structure determination. In particular, (1) self-rotation function calculations failed to yield clearly interpretable solutions, (2) the analysis of difference Patterson maps for heavy-atom derivatives required the development of a Patterson search program suite GROPAT. The redundancy in the asymmetric unit allowed refinement of poor-quality isomorphous phases at 4 A resolution and phase extension from 4 to 2.9 A resolution using real-space symmetry averaging and solvent flattening in the absence of any isomorphous phase information. Despite further difficulties caused by structural differences between the six independent copies of the monomer the resultant electron density map was of high quality and proved to be easily interpretable.

Jones EY, Stuart DI, Walker NP. 1990. The three-dimensional structure of tumour necrosis factor. Prog Clin Biol Res, 349 pp. 321-327.

Jones EY, Stuart DI, Walker NP. 1989. Structure of tumour necrosis factor. Nature, 338 (6212), pp. 225-228. | Show Abstract | Read more

Tumour necrosis factor is a trimeric molecule, each subunit of which consists of an antiparallel beta-sandwich. Individual subunits from the trimer by a novel edge-to-face packing of beta-sheets. A comparison of the subunit fold with that of other proteins reveals a remarkable similarity to the 'jelly-roll' structural motif characteristic of viral coat proteins.

Jones EY, Miller A. 1987. Structural models for the N- and C-terminal telopeptide regions of interstitial collagens. Biopolymers, 26 (4), pp. 463-480. | Read more

Miller A, Bradshaw J, Jones EY, Fraser RD, MacRae TP, Suzuki E. 1985. The structure of collagen. Ciba Found Symp, 114 pp. 65-79. | Show Abstract

A knowledge of the structure of collagen fibrils is important for any rational discussion of the occurrence and treatment of fibrosis. The different genetic types of collagen, and the structure of the triple-helical molecule as refined from X-ray fibre diffraction data, are described. The problem of determining molecular arrangement in native tissues is discussed. The various models proposed for the molecular arrangement of type I collagen are compared and an account is given of the quasi-hexagonal model. A detailed analysis of the X-ray diffraction patterns from native type I collagen fibres is used to provide a quantitative description of the quasi-hexagonal model. Parameters such as molecular positions, azimuthal orientation and axial shift can be estimated from the diffraction patterns. These parameters refer to the helix main-chain. Side-chain conformations can then be built in by molecular graphics and the predicted X-ray pattern for the complete model compared with the observed pattern.

Kong Y, Janssen BJ, Malinauskas T, Vangoor VR, Coles CH, Kaufmann R, Ni T, Gilbert RJ, Padilla-Parra S, Pasterkamp RJ, Jones EY. 2016. Structural Basis for Plexin Activation and Regulation. Neuron, 91 (3), pp. 548-560. | Show Abstract | Read more

Class A plexins (PlxnAs) act as semaphorin receptors and control diverse aspects of nervous system development and plasticity, ranging from axon guidance and neuron migration to synaptic organization. PlxnA signaling requires cytoplasmic domain dimerization, but extracellular regulation and activation mechanisms remain unclear. Here we present crystal structures of PlxnA (PlxnA1, PlxnA2, and PlxnA4) full ectodomains. Domains 1-9 form a ring-like conformation from which the C-terminal domain 10 points away. All our PlxnA ectodomain structures show autoinhibitory, intermolecular "head-to-stalk" (domain 1 to domain 4-5) interactions, which are confirmed by biophysical assays, live cell fluorescence microscopy, and cell-based and neuronal growth cone collapse assays. This work reveals a 2-fold role of the PlxnA ectodomains: imposing a pre-signaling autoinhibitory separation for the cytoplasmic domains via intermolecular head-to-stalk interactions and supporting dimerization-based PlxnA activation upon ligand binding. More generally, our data identify a novel molecular mechanism for preventing premature activation of axon guidance receptors.

Kakugawa S, Langton PF, Zebisch M, Howell S, Chang TH, Liu Y, Feizi T, Bineva G, O'Reilly N, Snijders AP et al. 2015. Notum deacylates Wnt proteins to suppress signalling activity. Nature, 519 (7542), pp. 187-192. | Show Abstract | Read more

Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase.

Chang TH, Hsieh FL, Zebisch M, Harlos K, Elegheert J, Jones EY. 2015. Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan. Elife, 4 (JULY 2015), pp. 1-27. | Show Abstract | Read more

Wnt signalling regulates multiple processes including angiogenesis, inflammation, and tumorigenesis. Norrin (Norrie Disease Protein) is a cystine-knot like growth factor. Although unrelated to Wnt, Norrin activates the Wnt/β-catenin pathway. Signal complex formation involves Frizzled4 (Fz4), low-density lipoprotein receptor related protein 5/6 (Lrp5/6), Tetraspanin-12 and glycosaminoglycans (GAGs). Here, we report crystallographic and small-angle X-ray scattering analyses of Norrin in complex with Fz4 cysteine-rich domain (Fz4CRD), of this complex bound with GAG analogues, and of unliganded Norrin and Fz4CRD. Our structural, biophysical and cellular data, map Fz4 and putative Lrp5/6 binding sites to distinct patches on Norrin, and reveal a GAG binding site spanning Norrin and Fz4CRD. These results explain numerous disease-associated mutations. Comparison with the Xenopus Wnt8-mouse Fz8CRD complex reveals Norrin mimics Wnt for Frizzled recognition. The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling.

Malinauskas T, Jones EY. 2014. Extracellular modulators of Wnt signalling. Curr Opin Struct Biol, 29 pp. 77-84. | Show Abstract | Read more

Wnt morphogens are secreted signalling proteins that play leading roles in embryogenesis and tissue homeostasis throughout life. Wnt signalling is controlled by multiple mechanisms, including posttranslational modification of Wnts, antagonist binding (to Wnts or their receptors), and regulation of the availability of Wnt receptors. Recent crystallographic, structure-guided biophysical and cell-based studies have advanced our understanding of how Wnt signalling is regulated at the cell surface. Structures include Wnt in complex with the cysteine-rich domain (CRD) of Frizzled, extracellular fragments of Wnt co-receptor LRP6, LRP6-binding antagonists Dickkopf and Sclerostin, antagonists 5T4/WAIF1 and Wnt inhibitory factor 1 (WIF-1), as well as Frizzled-ubiquitin ligases ZNRF3/RNF43 (in isolation and in complexes with Wnt signalling promoters R-spondins and LGR5). We review recent discoveries and remaining questions.

Seiradake E, del Toro D, Nagel D, Cop F, Härtl R, Ruff T, Seyit-Bremer G, Harlos K, Border EC, Acker-Palmer A et al. 2014. FLRT structure: balancing repulsion and cell adhesion in cortical and vascular development. Neuron, 84 (2), pp. 370-385. | Show Abstract | Read more

FLRTs are broadly expressed proteins with the unique property of acting as homophilic cell adhesion molecules and as heterophilic repulsive ligands of Unc5/Netrin receptors. How these functions direct cell behavior and the molecular mechanisms involved remain largely unclear. Here we use X-ray crystallography to reveal the distinct structural bases for FLRT-mediated cell adhesion and repulsion in neurons. We apply this knowledge to elucidate FLRT functions during cortical development. We show that FLRTs regulate both the radial migration of pyramidal neurons, as well as their tangential spread. Mechanistically, radial migration is controlled by repulsive FLRT2-Unc5D interactions, while spatial organization in the tangential axis involves adhesive FLRT-FLRT interactions. Further, we show that the fundamental mechanisms of FLRT adhesion and repulsion are conserved between neurons and vascular endothelial cells. Our results reveal FLRTs as powerful guidance factors with structurally encoded repulsive and adhesive surfaces.

Coles CH, Mitakidis N, Zhang P, Elegheert J, Lu W, Stoker AW, Nakagawa T, Craig AM, Jones EY, Aricescu AR. 2014. Structural basis for extracellular cis and trans RPTPσ signal competition in synaptogenesis. Nat Commun, 5 pp. 5209. | Show Abstract | Read more

Receptor protein tyrosine phosphatase sigma (RPTPσ) regulates neuronal extension and acts as a presynaptic nexus for multiple protein and proteoglycan interactions during synaptogenesis. Unknown mechanisms govern the shift in RPTPσ function, from outgrowth promotion to synaptic organization. Here, we report crystallographic, electron microscopic and small-angle X-ray scattering analyses, which reveal sufficient inter-domain flexibility in the RPTPσ extracellular region for interaction with both cis (same cell) and trans (opposite cell) ligands. Crystal structures of RPTPσ bound to its postsynaptic ligand TrkC detail an interaction surface partially overlapping the glycosaminoglycan-binding site. Accordingly, heparan sulphate and heparin oligomers compete with TrkC for RPTPσ binding in vitro and disrupt TrkC-dependent synaptic differentiation in neuronal co-culture assays. We propose that transient RPTPσ ectodomain emergence from the presynaptic proteoglycan layer allows capture by TrkC to form a trans-synaptic complex, the consequent reduction in RPTPσ flexibility potentiating interactions with additional ligands to orchestrate excitatory synapse formation.

Zebisch M, Xu Y, Krastev C, MacDonald BT, Chen M, Gilbert RJ, He X, Jones EY. 2013. Structural and molecular basis of ZNRF3/RNF43 transmembrane ubiquitin ligase inhibition by the Wnt agonist R-spondin. Nat Commun, 4 pp. 2787. | Show Abstract | Read more

The four R-spondin (Rspo) proteins are secreted agonists of Wnt signalling in vertebrates, functioning in embryogenesis and adult stem cell biology. Through ubiquitination and degradation of Wnt receptors, the transmembrane E3 ubiquitin ligase ZNRF3 and related RNF43 antagonize Wnt signalling. Rspo ligands have been reported to inhibit the ligase activity through direct interaction with ZNRF3 and RNF43. Here we report multiple crystal structures of the ZNRF3 ectodomain (ZNRF3(ecto)), a signalling-competent Furin1-Furin2 (Fu1-Fu2) fragment of Rspo2 (Rspo2(Fu1-Fu2)), and Rspo2(Fu1-Fu2) in complex with ZNRF3(ecto), or RNF43(ecto). A prominent loop in Fu1 clamps into equivalent grooves in the ZNRF3(ecto) and RNF43(ecto) surface. Rspo binding enhances dimerization of ZNRF3(ecto) but not of RNF43(ecto). Comparison of the four Rspo proteins, mutants and chimeras in biophysical and cellular assays shows that their signalling potency depends on their ability to recruit ZNRF3 or RNF43 via Fu1 into a complex with LGR receptors, which interact with Rspo via Fu2.

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Scopus

Seiradake E, Schaupp A, Del Toro Ruiz D, Kaufmann R, Mitakidis N, Harlos K, Aricescu AR, Klein R, Jones EY. 2013. Structurally encoded intraclass differences in EphA clusters drive distinct cell responses Nature Structural and Molecular Biology, 20 (8), pp. 958-964. | Show Abstract | Read more

Functional outcomes of ephrin binding to Eph receptors (Ephs) range from cell repulsion to adhesion. Here we used cell collapse and stripe assays, showing contrasting effects of human ephrinA5 binding to EphA2 and EphA4. Despite equivalent ligand binding affinities, EphA4 triggered greater cell collapse, whereas EphA2-expressing cells adhered better to ephrinA5-coated surfaces. Chimeric receptors showed that the ectodomain is a major determinant of cell response. We report crystal structures of EphA4 ectodomain alone and in complexes with ephrinB3 and ephrinA5. These revealed closed clusters with a dimeric or circular arrangement in the crystal lattice, contrasting with extended arrays previously observed for EphA2 ectodomain. Localization microscopy showed that ligand-stimulated EphA4 induces smaller clusters than does EphA2. Mutant Ephs link these characteristics to interactions observed in the crystal lattices, suggesting a mechanism by which distinctive ectodomain surfaces determine clustering, and thereby signaling, properties. © 2013 Nature America, Inc. All rights reserved.

Seiradake E, Schaupp A, del Toro Ruiz D, Kaufmann R, Mitakidis N, Harlos K, Aricescu AR, Klein R, Jones EY. 2013. Structurally encoded intraclass differences in EphA clusters drive distinct cell responses. Nat Struct Mol Biol, 20 (8), pp. 958-964. | Show Abstract | Read more

Functional outcomes of ephrin binding to Eph receptors (Ephs) range from cell repulsion to adhesion. Here we used cell collapse and stripe assays, showing contrasting effects of human ephrinA5 binding to EphA2 and EphA4. Despite equivalent ligand binding affinities, EphA4 triggered greater cell collapse, whereas EphA2-expressing cells adhered better to ephrinA5-coated surfaces. Chimeric receptors showed that the ectodomain is a major determinant of cell response. We report crystal structures of EphA4 ectodomain alone and in complexes with ephrinB3 and ephrinA5. These revealed closed clusters with a dimeric or circular arrangement in the crystal lattice, contrasting with extended arrays previously observed for EphA2 ectodomain. Localization microscopy showed that ligand-stimulated EphA4 induces smaller clusters than does EphA2. Mutant Ephs link these characteristics to interactions observed in the crystal lattices, suggesting a mechanism by which distinctive ectodomain surfaces determine clustering, and thereby signaling, properties.

Williams C, Hoppe HJ, Rezgui D, Strickland M, Forbes BE, Grutzner F, Frago S, Ellis RZ, Wattana-Amorn P, Prince SN et al. 2012. An exon splice enhancer primes IGF2:IGF2R binding site structure and function evolution. Science, 338 (6111), pp. 1209-1213. | Show Abstract | Read more

Placental development and genomic imprinting coevolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes IGF2 and IGF2R code for the growth promoter insulin-like growth factor 2 (IGF2) and its inhibitor, mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R), respectively. M6P/IGF2R of birds and fish do not recognize IGF2. In monotremes, which lack imprinting, IGF2 specifically bound M6P/IGF2R via a hydrophobic CD loop. We show that the DNA coding the CD loop in monotremes functions as an exon splice enhancer (ESE) and that structural evolution of binding site loops (AB, HI, FG) improved therian IGF2 affinity. We propose that ESE evolution led to the fortuitous acquisition of IGF2 binding by M6P/IGF2R that drew IGF2R into parental conflict; subsequent imprinting may then have accelerated affinity maturation.

Janssen BJ, Malinauskas T, Weir GA, Cader MZ, Siebold C, Jones EY. 2012. Neuropilins lock secreted semaphorins onto plexins in a ternary signaling complex. Nat Struct Mol Biol, 19 (12), pp. 1293-1299. | Show Abstract | Read more

Co-receptors add complexity to cell-cell signaling systems. The secreted semaphorin 3s (Sema3s) require a co-receptor, neuropilin (Nrp), to signal through plexin As (PlxnAs) in functions ranging from axon guidance to bone homeostasis, but the role of the co-receptor is obscure. Here we present the low-resolution crystal structure of a mouse semaphorin-plexin-Nrp complex alongside unliganded component structures. Dimeric semaphorin, two copies of plexin and two copies of Nrp are arranged as a dimer of heterotrimers. In each heterotrimer subcomplex, semaphorin contacts plexin, similar to in co-receptor-independent signaling complexes. The Nrp1s cross brace the assembly, bridging between sema domains of the Sema3A and PlxnA2 subunits from the two heterotrimers. Biophysical and cellular analyses confirm that this Nrp binding mode stabilizes a canonical, but weakened, Sema3-PlxnA interaction, adding co-receptor control over the mechanism by which receptor dimerization and/or oligomerization triggers signaling.

Chen S, Bubeck D, MacDonald BT, Liang WX, Mao JH, Malinauskas T, Llorca O, Aricescu AR, Siebold C, He X, Jones EY. 2011. Structural and functional studies of LRP6 ectodomain reveal a platform for Wnt signaling. Dev Cell, 21 (5), pp. 848-861. | Show Abstract | Read more

LDL-receptor-related protein 6 (LRP6), alongside Frizzled receptors, transduces Wnt signaling across the plasma membrane. The LRP6 ectodomain comprises four tandem β-propeller-EGF-like domain (PE) pairs that harbor binding sites for Wnt morphogens and their antagonists including Dickkopf 1 (Dkk1). To understand how these multiple interactions are integrated, we combined crystallographic analysis of the third and fourth PE pairs with electron microscopy (EM) to determine the complete ectodomain structure. An extensive inter-pair interface, conserved for the first-to-second and third-to-fourth PE interactions, contributes to a compact platform-like architecture, which is disrupted by mutations implicated in developmental diseases. EM reconstruction of the LRP6 platform bound to chaperone Mesd exemplifies a binding mode spanning PE pairs. Cellular and binding assays identify overlapping Wnt3a- and Dkk1-binding surfaces on the third PE pair, consistent with steric competition, but also suggest a model in which the platform structure supports an interplay of ligands through multiple interaction sites.

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