register interest

Professor A. Radu Aricescu

Research Area: Protein Science and Structural Biology
Technology Exchange: Bioinformatics, Crystallography, Drug discovery, Microscopy (Confocal) and Protein interaction
Scientific Themes: Protein Science & Structural Biology
Keywords: learning and memory, synaptic plasticity, glutamate receptor, GABA receptor, protein tyrosine phosphatase, protein tyrosine kinase and x-ray crystallography
Web Links:
Crystal structure of a human GABAA receptor (Miller & Aricescu, 2014).

Crystal structure of a human GABAA receptor (Miller & Aricescu, 2014).

Conformational plasticity of type IIa receptor protein tyrosine phosphatases facilitates proteoglycan binding (Coles at al, 2011).

Conformational plasticity of type IIa receptor protein tyrosine phosphatases facilitates ...

We are interested in understanding the molecular mechanisms responsible for acquisition and storage of information in the brain, i.e. learning and memory. Research over the past five decades led to the idea that activity-dependent changes in the strength of synapses between neurons may represent a mechanism for information storage. The neurotransmitters glutamate and gamma-amino-butyric acid (GABA) mediate most synaptic signalling in the vertebrate central nervous system. Physiologically, at synaptic sites, their receptors are incorporated into complex protein networks that span the inter-neuronal cleft and are essential for the in the induction, expression and/or modulation of synaptic plasticity. On the other hand, dysfunction of the same molecular systems appears to be responsible for the cognitive decline linked to the 'normal' aging process as well as a wide spectrum of neurological and psychiatric disorders including Alzheimer's, schizophrenia, Parkinson's, epilepsy, insomnia and major clinical depression.
Research in our group is currently focused on the structural and functional characterisation of synaptic receptors, our main tools being X-ray crystallography (to gain high resolution information) and increasingly electron microscopy (to study non-periodical, supra-molecular assemblies). We aim to understand principles governing the molecular organisation of neuronal synapses, and to devise ways of modulating synaptic function.

Name Department Institution Country
Professor Michisuke Yuzaki Keio University, School of Medicine Japan
Dr Ingo Greger MRC Laboratory of Molecular Biology, Cambridge United Kingdom
Professor Christian Siebold Structural Biology Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Professor Juha T Huiskonen Structural Biology Oxford University, Oxford Particle Imaging Centre United Kingdom
Dr Sergi Padilla-Parra Structural Biology Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Davies B, Brown LA, Cais O, Watson J, Clayton AJ, Chang VT, Biggs D, Preece C, Hernandez-Pliego P, Krohn J et al. 2017. A point mutation in the ion conduction pore of AMPA receptor GRIA3 causes dramatically perturbed sleep patterns as well as intellectual disability. Hum Mol Genet, 26 (20), pp. 3869-3882. | Show Abstract | Read more

The discovery of genetic variants influencing sleep patterns can shed light on the physiological processes underlying sleep. As part of a large clinical sequencing project, WGS500, we sequenced a family in which the two male children had severe developmental delay and a dramatically disturbed sleep-wake cycle, with very long wake and sleep durations, reaching up to 106-h awake and 48-h asleep. The most likely causal variant identified was a novel missense variant in the X-linked GRIA3 gene, which has been implicated in intellectual disability. GRIA3 encodes GluA3, a subunit of AMPA-type ionotropic glutamate receptors (AMPARs). The mutation (A653T) falls within the highly conserved transmembrane domain of the ion channel gate, immediately adjacent to the analogous residue in the Grid2 (glutamate receptor) gene, which is mutated in the mouse neurobehavioral mutant, Lurcher. In vitro, the GRIA3(A653T) mutation stabilizes the channel in a closed conformation, in contrast to Lurcher. We introduced the orthologous mutation into a mouse strain by CRISPR-Cas9 mutagenesis and found that hemizygous mutants displayed significant differences in the structure of their activity and sleep compared to wild-type littermates. Typically, mice are polyphasic, exhibiting multiple sleep bouts of sleep several minutes long within a 24-h period. The Gria3A653T mouse showed significantly fewer brief bouts of activity and sleep than the wild-types. Furthermore, Gria3A653T mice showed enhanced period lengthening under constant light compared to wild-type mice, suggesting an increased sensitivity to light. Our results suggest a role for GluA3 channel activity in the regulation of sleep behavior in both mice and humans.

Miller PS, Scott S, Masiulis S, De Colibus L, Pardon E, Steyaert J, Aricescu AR. 2017. Structural basis for GABAA receptor potentiation by neurosteroids. Nat Struct Mol Biol, | Show Abstract | Read more

Type A γ-aminobutyric acid receptors (GABAARs) are the principal mediators of inhibitory neurotransmission in the human brain. Endogenous neurosteroids interact with GABAARs to regulate acute and chronic anxiety and are potent sedative, analgesic, anticonvulsant and anesthetic agents. Their mode of binding and mechanism of receptor potentiation, however, remain unknown. Here we report crystal structures of a chimeric GABAAR construct in apo and pregnanolone-bound states. The neurosteroid-binding site is mechanically coupled to the helices lining the ion channel pore and modulates the desensitization-gate conformation. We demonstrate that the equivalent site is responsible for physiological, heteromeric GABAAR potentiation and explain the contrasting modulatory properties of 3a versus 3b neurosteroid epimers. These results illustrate how peripheral lipid ligands can regulate the desensitization gate of GABAARs, a process of broad relevance to pentameric ligand-gated ion channels.

Elegheert J, Cvetkovska V, Clayton AJ, Heroven C, Vennekens KM, Smukowski SN, Regan MC, Jia W, Smith AC, Furukawa H et al. 2017. Structural Mechanism for Modulation of Synaptic Neuroligin-Neurexin Signaling by MDGA Proteins. Neuron, 96 (1), pp. 242-244. | Read more

Elegheert J, Cvetkovska V, Clayton AJ, Heroven C, Vennekens KM, Smukowski SN, Regan MC, Jia W, Smith AC, Furukawa H et al. 2017. Structural Mechanism for Modulation of Synaptic Neuroligin-Neurexin Signaling by MDGA Proteins. Neuron, 95 (4), pp. 896-913.e10. | Show Abstract | Read more

Neuroligin-neurexin (NL-NRX) complexes are fundamental synaptic organizers in the central nervous system. An accurate spatial and temporal control of NL-NRX signaling is crucial to balance excitatory and inhibitory neurotransmission, and perturbations are linked with neurodevelopmental and psychiatric disorders. MDGA proteins bind NLs and control their function and interaction with NRXs via unknown mechanisms. Here, we report crystal structures of MDGA1, the NL1-MDGA1 complex, and a spliced NL1 isoform. Two large, multi-domain MDGA molecules fold into rigid triangular structures, cradling a dimeric NL to prevent NRX binding. Structural analyses guided the discovery of a broad, splicing-modulated interaction network between MDGA and NL family members and helped rationalize the impact of autism-linked mutations. We demonstrate that expression levels largely determine whether MDGAs act selectively or suppress the synapse organizing function of multiple NLs. These results illustrate a potentially brain-wide regulatory mechanism for NL-NRX signaling modulation.

Elegheert J, Kakegawa W, Clay JE, Shanks NF, Behiels E, Matsuda K, Kohda K, Miura E, Rossmann M, Mitakidis N et al. 2016. Structural basis for integration of GluD receptors within synaptic organizer complexes. Science, 353 (6296), pp. 295-299. | Show Abstract | Read more

Ionotropic glutamate receptor (iGluR) family members are integrated into supramolecular complexes that modulate their location and function at excitatory synapses. However, a lack of structural information beyond isolated receptors or fragments thereof currently limits the mechanistic understanding of physiological iGluR signaling. Here, we report structural and functional analyses of the prototypical molecular bridge linking postsynaptic iGluR δ2 (GluD2) and presynaptic β-neurexin 1 (β-NRX1) via Cbln1, a C1q-like synaptic organizer. We show how Cbln1 hexamers "anchor" GluD2 amino-terminal domain dimers to monomeric β-NRX1. This arrangement promotes synaptogenesis and is essential for D: -serine-dependent GluD2 signaling in vivo, which underlies long-term depression of cerebellar parallel fiber-Purkinje cell (PF-PC) synapses and motor coordination in developing mice. These results lead to a model where protein and small-molecule ligands synergistically control synaptic iGluR function.

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.

Matsuda K, Budisantoso T, Mitakidis N, Sugaya Y, Miura E, Kakegawa W, Yamasaki M, Konno K, Uchigashima M, Abe M et al. 2016. Transsynaptic Modulation of Kainate Receptor Functions by C1q-like Proteins. Neuron, 90 (4), pp. 752-767. | Show Abstract | Read more

Postsynaptic kainate-type glutamate receptors (KARs) regulate synaptic network activity through their slow channel kinetics, most prominently at mossy fiber (MF)-CA3 synapses in the hippocampus. Nevertheless, how KARs cluster and function at these synapses has been unclear. Here, we show that C1q-like proteins C1ql2 and C1ql3, produced by MFs, serve as extracellular organizers to recruit functional postsynaptic KAR complexes to the CA3 pyramidal neurons. C1ql2 and C1ql3 specifically bound the amino-terminal domains of postsynaptic GluK2 and GluK4 KAR subunits and the presynaptic neurexin 3 containing a specific sequence in vitro. In C1ql2/3 double-null mice, CA3 synaptic responses lost the slow, KAR-mediated components. Furthermore, despite induction of MF sprouting in a temporal lobe epilepsy model, KARs were not recruited to postsynaptic sites in C1ql2/3 double-null mice, leading to reduced recurrent circuit activities. C1q family proteins, broadly expressed, are likely to modulate KAR function throughout the brain and represent promising antiepileptic targets.

Yuzaki M, Aricescu AR. 2017. A GluD Coming-Of-Age Story. Trends Neurosci, 40 (3), pp. 138-150. | Show Abstract | Read more

The GluD1 and GluD2 receptors form the GluD ionotropic glutamate receptor (iGluR) subfamily. Without known endogenous ligands, they have long been referred to as 'orphan' and remained enigmatic functionally. Recent progress has, however, radically changed this view. Both GluD receptors are expressed in wider brain regions than originally thought. Human genetic studies and analyses of knockout mice have revealed their involvement in multiple neurodevelopmental and psychiatric disorders. The discovery of endogenous ligands, together with structural investigations, has opened the way towards a mechanistic understanding of GluD signaling at central nervous system synapses. These studies have also prompted the hypothesis that all iGluRs, and potentially other neurotransmitter receptors, rely on the cooperative binding of extracellular small-molecule and protein ligands for physiological signaling.

Gallimore AR, Aricescu AR, Yuzaki M, Calinescu R. 2016. A Computational Model for the AMPA Receptor Phosphorylation Master Switch Regulating Cerebellar Long-Term Depression. PLoS Comput Biol, 12 (1), pp. e1004664. | Show Abstract | Read more

The expression of long-term depression (LTD) in cerebellar Purkinje cells results from the internalisation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) from the postsynaptic membrane. This process is regulated by a complex signalling pathway involving sustained protein kinase C (PKC) activation, inhibition of serine/threonine phosphatase, and an active protein tyrosine phosphatase, PTPMEG. In addition, two AMPAR-interacting proteins-glutamate receptor-interacting protein (GRIP) and protein interacting with C kinase 1 (PICK1)-regulate the availability of AMPARs for trafficking between the postsynaptic membrane and the endosome. Here we present a new computational model of these overlapping signalling pathways. The model reveals how PTPMEG cooperates with PKC to drive LTD expression by facilitating the effect of PKC on the dissociation of AMPARs from GRIP and thus their availability for trafficking. Model simulations show that LTD expression is increased by serine/threonine phosphatase inhibition, and negatively regulated by Src-family tyrosine kinase activity, which restricts the dissociation of AMPARs from GRIP under basal conditions. We use the model to expose the dynamic balance between AMPAR internalisation and reinsertion, and the phosphorylation switch responsible for the perturbation of this balance and for the rapid plasticity initiation and regulation. Our model advances the understanding of PF-PC LTD regulation and induction, and provides a validated extensible platform for more detailed studies of this fundamental synaptic process.

Doody KM, Stanford SM, Svensson MND, Sacchetti C, Firestein GS, Aricescu AR, Bottini N. 2015. Characterizing the RPTP sigma Mediated Proteoglycan Switch As a Target for Rheumatoid Arthritis Therapy ARTHRITIS & RHEUMATOLOGY, 67

Doody KM, Stanford SM, Sacchetti C, Svensson MN, Coles CH, Mitakidis N, Kiosses WB, Bartok B, Fos C, Cory E et al. 2015. Targeting phosphatase-dependent proteoglycan switch for rheumatoid arthritis therapy. Sci Transl Med, 7 (288), pp. 288ra76. | Show Abstract | Read more

Despite the availability of several therapies for rheumatoid arthritis (RA) that target the immune system, a large number of RA patients fail to achieve remission. Joint-lining cells, called fibroblast-like synoviocytes (FLS), become activated during RA and mediate joint inflammation and destruction of cartilage and bone. We identify RPTPσ, a transmembrane tyrosine phosphatase, as a therapeutic target for FLS-directed therapy. RPTPσ is reciprocally regulated by interactions with chondroitin sulfate or heparan sulfate containing extracellular proteoglycans in a mechanism called the proteoglycan switch. We show that the proteoglycan switch regulates FLS function. Incubation of FLS with a proteoglycan-binding RPTPσ decoy protein inhibited cell invasiveness and attachment to cartilage by disrupting a constitutive interaction between RPTPσ and the heparan sulfate proteoglycan syndecan-4. RPTPσ mediated the effect of proteoglycans on FLS signaling by regulating the phosphorylation and cytoskeletal localization of ezrin. Furthermore, administration of the RPTPσ decoy protein ameliorated in vivo human FLS invasiveness and arthritis severity in the K/BxN serum transfer model of RA. Our data demonstrate that FLS are regulated by an RPTPσ-dependent proteoglycan switch in vivo, which can be targeted for RA therapy. We envision that therapies targeting the proteoglycan switch or its intracellular pathway in FLS could be effective as a monotherapy or in combination with currently available immune-targeted agents to improve control of disease activity in RA patients.

Taylor JC, Martin HC, Lise S, Broxholme J, Cazier JB, Rimmer A, Kanapin A, Lunter G, Fiddy S, Allan C et al. 2015. Factors influencing success of clinical genome sequencing across a broad spectrum of disorders. Nat Genet, 47 (7), pp. 717-726. | Show Abstract | Read more

To assess factors influencing the success of whole-genome sequencing for mainstream clinical diagnosis, we sequenced 217 individuals from 156 independent cases or families across a broad spectrum of disorders in whom previous screening had identified no pathogenic variants. We quantified the number of candidate variants identified using different strategies for variant calling, filtering, annotation and prioritization. We found that jointly calling variants across samples, filtering against both local and external databases, deploying multiple annotation tools and using familial transmission above biological plausibility contributed to accuracy. Overall, we identified disease-causing variants in 21% of cases, with the proportion increasing to 34% (23/68) for mendelian disorders and 57% (8/14) in family trios. We also discovered 32 potentially clinically actionable variants in 18 genes unrelated to the referral disorder, although only 4 were ultimately considered reportable. Our results demonstrate the value of genome sequencing for routine clinical diagnosis but also highlight many outstanding challenges.

Kakegawa W, Mitakidis N, Miura E, Abe M, Matsuda K, Takeo YH, Kohda K, Motohashi J, Takahashi A, Nagao S et al. 2015. Anterograde C1ql1 signaling is required in order to determine and maintain a single-winner climbing fiber in the mouse cerebellum. Neuron, 85 (2), pp. 316-329. | Show Abstract | Read more

Neuronal networks are dynamically modified by selective synapse pruning during development and adulthood. However, how certain connections win the competition with others and are subsequently maintained is not fully understood. Here, we show that C1ql1, a member of the C1q family of proteins, is provided by climbing fibers (CFs) and serves as a crucial anterograde signal to determine and maintain the single-winner CF in the mouse cerebellum throughout development and adulthood. C1ql1 specifically binds to the brain-specific angiogenesis inhibitor 3 (Bai3), which is a member of the cell-adhesion G-protein-coupled receptor family and expressed on postsynaptic Purkinje cells. C1ql1-Bai3 signaling is required for motor learning but not for gross motor performance or coordination. Because related family members of C1ql1 and Bai3 are expressed in various brain regions, the mechanism described here likely applies to synapse formation, maintenance, and function in multiple neuronal circuits essential for important brain functions.

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.

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.

Cited:

217

Scopus

Miller PS, Aricescu AR. 2014. Crystal structure of a human GABA<inf>A</inf> receptor Nature, 512 (7514), pp. 270-275. | Show Abstract | Read more

Type-A γ-aminobutyric acid receptors (GABA A Rs) are the principal mediators of rapid inhibitory synaptic transmission in the human brain. A decline in GABA A R signalling triggers hyperactive neurological disorders such as insomnia, anxiety and epilepsy. Here we present the first three-dimensional structure of a GABA A R, the human β homopentamer, at 3Å resolution. This structure reveals architectural elements unique to eukaryotic Cys-loop receptors, explains the mechanistic consequences of multiple human disease mutations and shows an unexpected structural role for a conserved N-linked glycan. The receptor was crystallized bound to a previously unknown agonist, benzamidine, opening a new avenue for the rational design of GAB A A R modulators. The channel region forms a closed gate at the base of the pore, representative of a desensitized state. These results offer new insights into the signalling mechanisms of pentameric ligand-gated ion channels and enhance current understanding of GABAergic neurotransmission. © 2014 Macmillan Publishers Limited. All rights reserved.

Miller PS, Aricescu AR. 2014. Crystal structure of a human GABAA receptor. Nature, 512 (7514), pp. 270-275. | Show Abstract | Read more

Type-A γ-aminobutyric acid receptors (GABAARs) are the principal mediators of rapid inhibitory synaptic transmission in the human brain. A decline in GABAAR signalling triggers hyperactive neurological disorders such as insomnia, anxiety and epilepsy. Here we present the first three-dimensional structure of a GABAAR, the human β3 homopentamer, at 3 Å resolution. This structure reveals architectural elements unique to eukaryotic Cys-loop receptors, explains the mechanistic consequences of multiple human disease mutations and shows an unexpected structural role for a conserved N-linked glycan. The receptor was crystallized bound to a previously unknown agonist, benzamidine, opening a new avenue for the rational design of GABAAR modulators. The channel region forms a closed gate at the base of the pore, representative of a desensitized state. These results offer new insights into the signalling mechanisms of pentameric ligand-gated ion channels and enhance current understanding of GABAergic neurotransmission.

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.

Cited:

36

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.

Cited:

25

Scopus

Aricescu AR, Owens RJ. 2013. Expression of recombinant glycoproteins in mammalian cells: Towards an integrative approach to structural biology Current Opinion in Structural Biology, 23 (3), pp. 345-356. | Show Abstract | Read more

Mammalian cells are rapidly becoming the system of choice for the production of recombinant glycoproteins for structural biology applications. Their use has enabled the structural investigation of a whole new set of targets including large, multi-domain and highly glycosylated eukaryotic cell surface receptors and their supra-molecular assemblies. We summarize the technical advances that have been made in mammalian expression technology and highlight some of the structural insights that have been obtained using these methods. Looking forward, it is clear that mammalian cell expression will provide exciting and unique opportunities for an integrative approach to the structural study of proteins, especially of human origin and medically relevant, by bridging the gap between the purified state and the cellular context. © 2013 Elsevier Ltd.

Cited:

67

Scopus

Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR. 2013. Atomic-resolution monitoring of protein maturation in live human cells by NMR Nature Chemical Biology, 9 (5), pp. 297-299. | Show Abstract | Read more

We use NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1). We follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 oxidizes the SOD1 intrasubunit disulfide bond through both copper-dependent and copper-independent mechanisms. Our approach represents a new strategy for structural investigation of endogenously expressed proteins in a physiological (cellular) environment. © 2013 Nature America, Inc.

Aricescu AR, Owens RJ. 2013. Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology. Curr Opin Struct Biol, 23 (3), pp. 345-356. | Show Abstract | Read more

Mammalian cells are rapidly becoming the system of choice for the production of recombinant glycoproteins for structural biology applications. Their use has enabled the structural investigation of a whole new set of targets including large, multi-domain and highly glycosylated eukaryotic cell surface receptors and their supra-molecular assemblies. We summarize the technical advances that have been made in mammalian expression technology and highlight some of the structural insights that have been obtained using these methods. Looking forward, it is clear that mammalian cell expression will provide exciting and unique opportunities for an integrative approach to the structural study of proteins, especially of human origin and medically relevant, by bridging the gap between the purified state and the cellular context.

Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR. 2013. Atomic-resolution monitoring of protein maturation in live human cells by NMR. Nat Chem Biol, 9 (5), pp. 297-299. | Show Abstract | Read more

We use NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1). We follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 oxidizes the SOD1 intrasubunit disulfide bond through both copper-dependent and copper-independent mechanisms. Our approach represents a new strategy for structural investigation of endogenously expressed proteins in a physiological (cellular) environment.

Bell CH, Healey E, van Erp S, Bishop B, Tang C, Gilbert RJC, Aricescu AR, Pasterkamp RJ, Siebold C. 2013. Structure of the repulsive guidance molecule (RGM)-neogenin signaling hub. Science, 341 (6141), pp. 77-80. | Show Abstract | Read more

Repulsive guidance molecule family members (RGMs) control fundamental and diverse cellular processes, including motility and adhesion, immune cell regulation, and systemic iron metabolism. However, it is not known how RGMs initiate signaling through their common cell-surface receptor, neogenin (NEO1). Here, we present crystal structures of the NEO1 RGM-binding region and its complex with human RGMB (also called dragon). The RGMB structure reveals a previously unknown protein fold and a functionally important autocatalytic cleavage mechanism and provides a framework to explain numerous disease-linked mutations in RGMs. In the complex, two RGMB ectodomains conformationally stabilize the juxtamembrane regions of two NEO1 receptors in a pH-dependent manner. We demonstrate that all RGM-NEO1 complexes share this architecture, which therefore represents the core of multiple signaling pathways.

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.

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.

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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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.

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.

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.

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 (12), pp. 1679. | Read more

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.

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.

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.

Staunton D, Millard CJ, Aricescu AR, Campbell ID. 2009. Preparation of recombinant fibronectin fragments for functional and structural studies. Methods Mol Biol, 522 pp. 73-99. | Show Abstract | Read more

Fibronectin, an ubiquitous extracellular matrix (ECM) glycoprotein, plays a major role in fundamental biological processes such as cell adhesion and migration, maintenance of normal cell morphology, cytoskeletal organization, and cell differentiation. Fibronectin is constructed from three types of independently folding protein module (Fn1, Fn2, and Fn3) and is found as a Fibrillar network in the ECM where it interacts with other ECM components and provides anchorage sites for cell surface integrin receptors. The mosaic nature of fibronectin permits it to be analyzed by a "dissection" strategy, where protein fragments generated by recombinant expression in E. coli, P. pastoris, and human cell lines are employed in structural and functional investigations. We describe methods suitable for the production of various fibronectin fragments for study by a variety of techniques including crystallography and electron microscopy but special mention is made of methods suitable for the production of samples for NMR studies.

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.

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.

Maretto S, Müller PS, Aricescu AR, Cho KW, Bikoff EK, Robertson EJ. 2008. Ventral closure, headfold fusion and definitive endoderm migration defects in mouse embryos lacking the fibronectin leucine-rich transmembrane protein FLRT3. Dev Biol, 318 (1), pp. 184-193. | Show Abstract | Read more

The three fibronectin leucine-rich repeat transmembrane (FLRT) proteins contain 10 leucine-rich repeats (LRR), a type III fibronectin (FN) domain, followed by the transmembrane region, and a short cytoplasmic tail. XFLRT3, a Nodal/TGFbeta target, regulates cell adhesion and modulates FGF signalling during Xenopus gastrulation. The present study describes the onset and pattern of FLRT1-3 expression in the early mouse embryo. FLRT3 expression is activated in the anterior visceral endoderm (AVE), and during gastrulation appears in anterior streak derivatives namely the node, notochord and the emerging definitive endoderm. To explore FLRT3 function we generated a null allele via gene targeting. Early Nodal activities required for anterior-posterior (A-P) patterning, primitive streak formation and left-right (L-R) axis determination were unperturbed. However, FLRT3 mutant embryos display defects in headfold fusion, definitive endoderm migration and a failure of the lateral edges of the ventral body wall to fuse, leading to cardia bifida. Surprisingly, the mutation has no effect on FGF signalling. Collectively these experiments demonstrate that FLRT3 plays a key role in controlling cell adhesion and tissue morphogenesis in the developing mouse embryo.

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.

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.

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.

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.

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.

Nettleship JE, Aplin R, Aricescu AR, Evans EJ, Davis SJ, Crispin M, Owens RJ. 2007. Analysis of variable N-glycosylation site occupancy in glycoproteins by liquid chromatography electrospray ionization mass spectrometry. Anal Biochem, 361 (1), pp. 149-151. | Read more

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.

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.

Meier C, Aricescu AR, Assenberg R, Aplin RT, Gilbert RJ, Grimes JM, Stuart DI. 2006. The crystal structure of ORF-9b, a lipid binding protein from the SARS coronavirus. Structure, 14 (7), pp. 1157-1165. | Show Abstract | Read more

To achieve the greatest output from their limited genomes, viruses frequently make use of alternative open reading frames, in which translation is initiated from a start codon within an existing gene and, being out of frame, gives rise to a distinct protein product. These alternative protein products are, as yet, poorly characterized structurally. Here we report the crystal structure of ORF-9b, an alternative open reading frame within the nucleocapsid (N) gene from the SARS coronavirus. The protein has a novel fold, a dimeric tent-like beta structure with an amphipathic surface, and a central hydrophobic cavity that binds lipid molecules. This cavity is likely to be involved in membrane attachment and, in mammalian cells, ORF-9b associates with intracellular vesicles, consistent with a role in the assembly of the virion. Analysis of ORF-9b and other overlapping genes suggests that they provide snapshots of the early evolution of novel protein folds.

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.

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.

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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.

Sajnani-Perez G, Chilton JK, Aricescu AR, Haj F, Stoker AW. 2003. Isoform-specific binding of the tyrosine phosphatase PTPsigma to a ligand in developing muscle. Mol Cell Neurosci, 22 (1), pp. 37-48. | Show Abstract | Read more

PTPsigma is a receptor tyrosine phosphatase that is expressed widely in the developing nervous system and that controls the growth and retinotopic mapping of retinal axons. PTPsigma is also expressed in motor neurons where its function is unclear. Given that invertebrate relatives of PTPsigma can control motor axon guidance, target contact, and synaptogenesis, we have asked if extracellular ligands exist for cPTPsigma, the avian PTPsigma orthologue, in the neuromuscular system. Of the two major isoforms cPTPsigma1 and cPTPsigma2, only the shorter cPTPsigma1 isoform is expressed in developing spinal motor neurons and their axons. We show that ectodomains of cPTPsigma1, but not of cPTPsigma2, bind specifically to developing skeletal myotubes. The putative myotube ligand is not related to the previously described binding of cPTPsigma to heparan sulfates within the proteoglycans agrin and collagen XVIII, since heparinase treatment of myotubes does not alter cPTPsigma1 binding and since most mutations that abolish binding of cPTPsigma1 to heparin do not affect myotube binding. The expression of cPTPsigma1 in motor axons and its direct binding to target myotubes suggest an isoform-specific role for axonally expressed cPTPsigma1 during establishment or maintenance of neuromuscular contacts.

Rashid-Doubell F, McKinnell I, Aricescu AR, Sajnani G, Stoker A. 2002. Chick PTPsigma regulates the targeting of retinal axons within the optic tectum. J Neurosci, 22 (12), pp. 5024-5033. | Show Abstract

Chick PTPsigma (cPTPsigma), also known as CRYPalpha, is a receptor-like protein tyrosine phosphatase found on axons and growth cones. Putative ligands for cPTPsigma are distributed within basement membranes and on glial end feet of the retina, optic nerve, and optic tectum, suggesting that cPTPsigma signaling is occurring along the whole retinotectal pathway. We have shown previously that cPTPsigma plays a role in supporting the retinal phase of axon outgrowth. Here we have now addressed the role of cPTPsigma within retinal axons as they undergo growth and topographic targeting in the optic tectum. With the use of retroviruses, a secretable cPTPsigma ectodomain was ectopically expressed in ovo in the developing chick optic tectum, with the aim of directly disrupting the function of endogenous cPTPsigma. In ovo, the secreted ectodomains accumulated at tectal sites in which cPTPsigma ligands are also specifically found, suggesting that they are binding to these endogenous ligands. Anterograde labeling of retinal axons entering these optic tecta revealed abnormal axonal phenotypes. These included the premature stalling and arborization of fibers, excessive pretectal arbor formation, and diffuse termination zones. Most of the defects were rostral of the predicted termination zone, indicating that cPTPsigma function is necessary for sustaining the growth of retinal axons over the optic tectum and for directing axons to their correct sites of termination. This demonstrates that regulation of cPTPsigma signaling in retinal axons is required for their topographic mapping, the first evidence of this function for a receptor-like protein tyrosine phosphatase in the retinotectal projection.

Aricescu AR, McKinnell IW, Halfter W, Stoker AW. 2002. Heparan sulfate proteoglycans are ligands for receptor protein tyrosine phosphatase sigma. Mol Cell Biol, 22 (6), pp. 1881-1892. | Show Abstract | Read more

RPTPsigma is a cell adhesion molecule-like receptor protein tyrosine phosphatase involved in nervous system development. Its avian orthologue, known as cPTPsigma or CRYPalpha, promotes intraretinal axon growth and controls the morphology of growth cones. The molecular mechanisms underlying the functions of cPTPsigma are still to be determined, since neither its physiological ligand(s) nor its substrates have been described. Nevertheless, a major class of ligand(s) is present in the retinal basal lamina and glial endfeet, the potent native growth substrate for retinal axons. We demonstrate here that cPTPsigma is a heparin-binding protein and that its basal lamina ligands include the heparan sulfate proteoglycans (HSPGs) agrin and collagen XVIII. These molecules interact with high affinity with cPTPsigma in vitro, and this binding is totally dependent upon their heparan sulfate chains. Using molecular modelling and site-directed mutagenesis, a binding site for heparin and heparan sulfate was identified in the first immunoglobulin-like domain of cPTPsigma. HSPGs are therefore a novel class of heterotypic ligand for cPTPsigma, suggesting that cPTPsigma signaling in axons and growth cones is directly responsive to matrix-associated cues.

Szedlacsek SE, Aricescu AR, Fulga TA, Renault L, Scheidig AJ. 2001. Crystal structure of PTP-SL/PTPBR7 catalytic domain: implications for MAP kinase regulation. J Mol Biol, 311 (3), pp. 557-568. | Show Abstract | Read more

Protein tyrosine phosphatases PTP-SL and PTPBR7 are isoforms belonging to cytosolic membrane-associated and to receptor-like PTPs (RPTPs), respectively. They represent a new family of PTPs with a major role in activation and translocation of MAP kinases. Specifically, the complex formation between PTP-SL and ERK2 involves an unusual interaction leading to the phosphorylation of PTP-SL by ERK2 at Thr253 and the inactivating dephosphorylation of ERK2 by PTP-SL. This interaction is strictly dependent upon a kinase interaction motif (KIM) (residues 224-239) situated at the N terminus of the PTP-SL catalytic domain. We report the first crystal structure of the catalytic domain for a member of this family (PTP-SL, residues 254-549, identical with residues 361-656 of PTPBR7), providing an example of an RPTP with single cytoplasmic domain, which is monomeric, having an unhindered catalytic site. In addition to the characteristic PTP-core structure, PTP-SL has an N-terminal helix, possibly orienting the KIM motif upon interaction with the target ERK2. An unusual residue in the catalytically important WPD loop promotes formation of a hydrophobically and electrostatically stabilised clamp. This could induce increased rigidity to the WPD loop and therefore reduced catalytic activity, in agreement with our kinetic measurements. A docking model based on the PTP-SL structure suggests that, in the complex with ERK2, the phosphorylation of PTP-SL should be accomplished first. The subsequent dephosphorylation of ERK2 seems to be possible only if a conformational rearrangement of the two interacting partners takes place.

Aricescu AR, Fulga TA, Cismasiu V, Goody RS, Szedlacsek SE. 2001. Intramolecular interactions in protein tyrosine phosphatase RPTPmu: kinetic evidence. Biochem Biophys Res Commun, 280 (1), pp. 319-327. | Show Abstract | Read more

The receptor-like protein tyrosine phosphatase RPTPmu contains three intracellular domains: the juxtamembrane (JM) and two phosphatase domains (D1 and D2). D1 is catalytically active in vitro. The functional roles of JM and D2 are still unclear. To find out whether and how they modulate the phosphatase activity of D1, we compared the enzymatic characteristics of two constructs, containing a truncated JM and either D1 or both phosphatase domains. p-Nitrophenyl phosphate and two peptide substrates were efficiently dephosphorylated by both constructs. The specificity constant of D1 alone was up to 50% higher. D2 induces (a) decreased K(m) values for peptide substrates, (b) decreased catalytic efficiency for these substrates, (c) shifting of the optimal pH to slightly lower values, and (d) looser binding of competitive inhibitors. These data suggest that the phosphatase activity of D1 is negatively modulated and its ligand binding capacity is sensibly modified by domain D2, having possible functional significance.

Szedlacsek SE, Aricescu AR, Havsteen BH. 1996. Time-dependent control of metabolic systems by external effectors. J Theor Biol, 182 (3), pp. 341-350. | Show Abstract | Read more

The expression of elasticity coefficients for time-dependent enzyme inhibition/activation by an external effector was initially derived. Only a limited number of restrictive assumptions were used, for example, the enzyme was considered to obey Michaelis-Menten kinetics and effectors were taken to be competitive. Then, a simple metabolic system under the control of a time-dependent effector (inhibitor or activator) was analysed and the expressions of the control coefficients were obtained. In addition, two numerical examples were used to represent the control coefficients as functions of time and effector concentration. The results indicate that the control coefficients vary in a relatively limited range of values; however, for certain intervals of time and of effector concentration local minima or major modifications of the coefficients may be recorded. The physiological importance of non-steady state analysis of metabolic systems controlled by external effectors was also discussed. It was stressed that the non-steady state treatment may contribute to creating a more realistic image of the metabolic control processes.

Elegheert J, Kakegawa W, Clay JE, Shanks NF, Behiels E, Matsuda K, Kohda K, Miura E, Rossmann M, Mitakidis N et al. 2016. Structural basis for integration of GluD receptors within synaptic organizer complexes. Science, 353 (6296), pp. 295-299. | Show Abstract | Read more

Ionotropic glutamate receptor (iGluR) family members are integrated into supramolecular complexes that modulate their location and function at excitatory synapses. However, a lack of structural information beyond isolated receptors or fragments thereof currently limits the mechanistic understanding of physiological iGluR signaling. Here, we report structural and functional analyses of the prototypical molecular bridge linking postsynaptic iGluR δ2 (GluD2) and presynaptic β-neurexin 1 (β-NRX1) via Cbln1, a C1q-like synaptic organizer. We show how Cbln1 hexamers "anchor" GluD2 amino-terminal domain dimers to monomeric β-NRX1. This arrangement promotes synaptogenesis and is essential for D: -serine-dependent GluD2 signaling in vivo, which underlies long-term depression of cerebellar parallel fiber-Purkinje cell (PF-PC) synapses and motor coordination in developing mice. These results lead to a model where protein and small-molecule ligands synergistically control synaptic iGluR function.

Matsuda K, Budisantoso T, Mitakidis N, Sugaya Y, Miura E, Kakegawa W, Yamasaki M, Konno K, Uchigashima M, Abe M et al. 2016. Transsynaptic Modulation of Kainate Receptor Functions by C1q-like Proteins. Neuron, 90 (4), pp. 752-767. | Show Abstract | Read more

Postsynaptic kainate-type glutamate receptors (KARs) regulate synaptic network activity through their slow channel kinetics, most prominently at mossy fiber (MF)-CA3 synapses in the hippocampus. Nevertheless, how KARs cluster and function at these synapses has been unclear. Here, we show that C1q-like proteins C1ql2 and C1ql3, produced by MFs, serve as extracellular organizers to recruit functional postsynaptic KAR complexes to the CA3 pyramidal neurons. C1ql2 and C1ql3 specifically bound the amino-terminal domains of postsynaptic GluK2 and GluK4 KAR subunits and the presynaptic neurexin 3 containing a specific sequence in vitro. In C1ql2/3 double-null mice, CA3 synaptic responses lost the slow, KAR-mediated components. Furthermore, despite induction of MF sprouting in a temporal lobe epilepsy model, KARs were not recruited to postsynaptic sites in C1ql2/3 double-null mice, leading to reduced recurrent circuit activities. C1q family proteins, broadly expressed, are likely to modulate KAR function throughout the brain and represent promising antiepileptic targets.

Cited:

217

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Miller PS, Aricescu AR. 2014. Crystal structure of a human GABA<inf>A</inf> receptor Nature, 512 (7514), pp. 270-275. | Show Abstract | Read more

Type-A γ-aminobutyric acid receptors (GABA A Rs) are the principal mediators of rapid inhibitory synaptic transmission in the human brain. A decline in GABA A R signalling triggers hyperactive neurological disorders such as insomnia, anxiety and epilepsy. Here we present the first three-dimensional structure of a GABA A R, the human β homopentamer, at 3Å resolution. This structure reveals architectural elements unique to eukaryotic Cys-loop receptors, explains the mechanistic consequences of multiple human disease mutations and shows an unexpected structural role for a conserved N-linked glycan. The receptor was crystallized bound to a previously unknown agonist, benzamidine, opening a new avenue for the rational design of GAB A A R modulators. The channel region forms a closed gate at the base of the pore, representative of a desensitized state. These results offer new insights into the signalling mechanisms of pentameric ligand-gated ion channels and enhance current understanding of GABAergic neurotransmission. © 2014 Macmillan Publishers Limited. All rights reserved.

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.

Cited:

67

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Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR. 2013. Atomic-resolution monitoring of protein maturation in live human cells by NMR Nature Chemical Biology, 9 (5), pp. 297-299. | Show Abstract | Read more

We use NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1). We follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 oxidizes the SOD1 intrasubunit disulfide bond through both copper-dependent and copper-independent mechanisms. Our approach represents a new strategy for structural investigation of endogenously expressed proteins in a physiological (cellular) environment. © 2013 Nature America, Inc.

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.

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