Structural biology of cell surface receptors.

Project Overview

Prof E. Yvonne Jones

Structural basis of semaphorin-plexin signalling. Janssen et al., Nature (2010)

Notum deacylates Wnts to suppress signalling activity. Kakugawa et al., Nature (2015)

Many major questions in receptor biology centre on how interactions occurring outside the cell trigger signalling inside the cell. To answer such questions for the biomedically relevant systems used in human cells we need to be able to express, purify and structurally analyse membrane spanning glycoproteins. Many of the necessary methodologies have only very recently been developed, and these are now bearing fruit, so this is a very exciting time for the field of receptor research. We are one of relatively few structural biology laboratories worldwide with very substantial expertise in eukaryotic expression systems combined with access to methodologies ranging from protein crystallography and cryoEM to super-resolution light microscopy. My group provides opportunities for doctoral students who wish to use these facilities to undertake research into the detailed molecular mechanisms of cell surface receptor signalling systems. Current work underway in my laboratory focuses on macromolecular systems mediating cell-to-cell interactions which are of fundamental importance during embryogenesis as well as in adult tissue homeostasis. These studies follow two major themes:

  1. mechanisms determining cell guidance and adhesion
  2. mechanisms involved in the modulation of Wnt signalling

A flavour of our work on the extracellular side of the cell surface can be gained from recent publications (Kong et al Neuron 2016;  Kakugawa et al Nature 2015), current work in the laboratory is now heading into the membrane and involves a spectrum of techniques which seamlessly span from the atomic to the cellular scale.

Training Opportunities

The core technique in my laboratory has been x-ray crystallography, however, we are now also using cryoEM and graduate students in the lab will have the opportunity to gain experience of both techniques. As our aim is to generate insights which span from the detailed atomic structure to the cellular context doctoral students have access to a broad range of methodologies. These include molecular biology, prokaryotic and eukaryotic expression systems (including tissue culture of insect and mammalian cells), protein purification, biophysical techniques (including surface plasmon resonance and analytical ultracentrifugation), crystallization, sychrotron data collection, in silico structural analysis, super-resolution light and electron cryo microscopy. In addition we have extensive networks of interdisciplinary collaborations within Oxford as well as internationally so that research students can interface their results with the broader biomedical context (including when appropriate translational research). Doctoral students have joined the group after undergraduate studies in subjects ranging from clinical medicine to biochemistry, chemistry and physics. All share a passion for adding to our understanding of the molecular mechanisms which underpin biomedical research. The stories of Dr Tao-Hsin Chang, Dr Tomas Malinauskas, Dr Christian Bell, Dr Charlotte Coles and Dr Thomas Bowden, five ex-doctoral students who studied in my group, can be read in the student profiles section of this site. After his doctoral studies with me Tao-Hsin was awarded a Human Frontiers Science Programme (HFSP) Fellowship to continue his research career at Johns Hopkins University in the US. Similarly Tomas and Charlotte were awarded HFSP Fellowships to pursue postdoctoral studies in the US and Germany respectively. Christian is now a Section Head at Roche in Switzerland and Thomas is back at the University of Oxford leading his own research group as an Associate Professor and MRC Career Development Award Fellow.

Theme

Protein Science & Structural Biology and Cancer Biology

Admissions

Project reference number: 114

Funding and admissions information

Supervisors

Name Department Institution Country Email
Professor E. Yvonne Jones FRS FMedSci Structural Biology Oxford University, Henry Wellcome Building of Genomic Medicine GBR jones-pa@strubi.ox.ac.uk
Professor David Stuart FRS Structural Biology Oxford University, Henry Wellcome Building of Genomic Medicine GBR stuart-pa@strubi.ox.ac.uk

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. 548-60. Read 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. Hide abstract

Seiradake E, Jones EY, Klein R. 2016. Structural Perspectives on Axon Guidance. Annu. Rev. Cell Dev. Biol., 32 pp. 577-608. Read 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. Hide abstract

Kakugawa S, Langton PF, Zebisch M, Howell SA, Chang TH, Liu Y, Feizi T, Bineva G, O'Reilly N, Snijders AP, Jones EY, Vincent JP. 2015. Notum deacylates Wnt proteins to suppress signalling activity. Nature, 519 (7542), pp. 187-92. Read 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. Hide abstract

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 Read 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. Hide abstract

Seiradake E, del Toro D, Nagel D, Cop F, Härtl R, Ruff T, Seyit-Bremer G, Harlos K, Border EC, Acker-Palmer A, Jones EY, Klein R. 2014. FLRT structure: balancing repulsion and cell adhesion in cortical and vascular development. Neuron, 84 (2), pp. 370-85. Read 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. Hide abstract

Malinauskas T, Jones EY. 2014. Extracellular modulators of Wnt signalling. Curr. Opin. Struct. Biol., 29 pp. 77-84. Read 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. Hide abstract

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. 958-64. Read 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. Hide abstract

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. 1293-9. Read 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. Hide abstract

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. 848-61. Read 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. Hide abstract

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. 886-93. Read 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. Hide abstract

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. 484-8. Read 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. Hide abstract

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. 1118-22. Read 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. Hide abstract