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CD47 is the only 5-transmembrane (5-TM) spanning receptor of the immune system. Its extracellular domain (ECD) is a cell surface marker of self that binds SIRPα and inhibits macrophage phagocytosis, and cancer immuno-therapy approaches in clinical trials are focused on blocking CD47/SIRPα interaction. We present the crystal structure of full length CD47 bound to the function-blocking antibody B6H12. CD47 ECD is tethered to the TM domain via a six-residue peptide linker ( 114 RVVSWF 119 ) that forms an extended loop (SWF loop), with the fundamental role of inserting the side chains of W118 and F119 into the core of CD47 extracellular loop region (ECLR). Using hydrogen-deuterium exchange and molecular dynamics simulations we show that CD47’s ECLR architecture, comprised of two extracellular loops and the SWF loop, creates a molecular environment stabilizing the ECD for presentation on the cell surface. These findings provide insights into CD47 immune recognition, signaling and therapeutic intervention. CD47 is a transmembrane receptor involved in the regulation of various signalling pathways and a promising target for immuno-oncology therapeutics. Here, the authors present the crystal structure of full-length human CD47 and provide insights into the molecular mechanism of CD47-mediated signalling.

The CCR5 chemokine receptor acts as a co-receptor for HIV-1 viral entry. Here we report the 2.7 angstrom-resolution crystal structure of human CCR5 bound to the marketed HIV drug maraviroc. The structure reveals a ligand-binding site that is distinct from the proposed major recognition sites for chemokines and the viral glycoprotein gp120, providing insights into the mechanism of allosteric inhibition of chemokine signaling and viral entry. A comparison between CCR5 and CXCR4 crystal structures, along with models of co-receptor—gp120-V3 complexes, suggests that different charge distributions and steric hindrances caused by residue substitutions may be major determinants of HIV-1 co-receptor selectivity. These high-resolution insights into CCR5 can enable structure-based drug discovery for the treatment of HIV-1 infection.

The enzyme glutamate decarboxylase (GAD) produces the neurotransmitter GABA, using pyridoxal-5’-phosphate (PLP). GAD exists as two isoforms, GAD65 and GAD67. Only GAD65 acts as a major autoantigen, frequently implicated in type 1 diabetes and other autoimmune diseases. Here we characterize the structure and dynamics of GAD65 and its interaction with the autoimmune polyendocrine syndrome type 2-associated autoantibody b96.11. Using hydrogen-deuterium exchange mass spectrometry (HDX), X-ray crystallography, cryo-electron microscopy, and computational approaches, we examine the conformational dynamics of apo- and holoGAD65 and the GAD65-autoantibody complex. HDX reveals local dynamics accompanying autoinactivation, with the catalytic loop promoting collective motions at the CTD-PLP domain interface. In the GAD65-b96.11 complex, heavy chain CDRs dominate the interaction, with a long CDRH3 bridging the GAD65 dimer via electrostatic interactions with the 260 PEVKEK 265 motif. This bridging links structural elements controlling GAD65’s conformational flexibility to its autoantigenicity. Thus, intrinsic dynamics, rather than sequence differences within epitopes, appear to be responsible for the contrasting autoantigenicities of GAD65 and GAD67. Our findings elucidate the structural and dynamic factors that govern the varying autoantibody reactivities of GAD65 and GAD67, offering a revised rationale for the autoimmune response to GAD65. Using HDX-MS, X-ray crystallography, cryo-EM, and MD simulations, the authors examined the conformational dynamics of GAD65 in its apo- and holo- states and its interaction with the autoimmune polyendocrine syndrome type 2-associated autoantibody b96.11.

Gamma-aminobutyric acid (GABA) is synthesized by two isoforms of the pyridoxal 5′-phosphate–dependent enzyme glutamic acid decarboxylase (GAD65 and GAD67). GAD67 is constitutively active and is responsible for basal GABA production. In contrast, GAD65, an autoantigen in type I diabetes, is transiently activated in response to the demand for extra GABA in neurotransmission, and cycles between an active holo form and an inactive apo form. We have determined the crystal structures of N-terminal truncations of both GAD isoforms. The structure of GAD67 shows a tethered loop covering the active site, providing a catalytic environment that sustains GABA production. In contrast, the same catalytic loop is inherently mobile in GAD65. Kinetic studies suggest that mobility in the catalytic loop promotes a side reaction that results in cofactor release and GAD65 autoinactivation. These data reveal the molecular basis for regulation of GABA homeostasis.

Chemokines and their receptors control cell migration during development, immune system responses, and in numerous diseases, including inflammation and cancer. The structural basis of receptor:chemokine recognition has been a long-standing unanswered question due to the challenges of structure determination for membrane protein complexes. Here, we report the crystal structure of the chemokine receptor CXCR4 in complex with the viral chemokine antagonist vMIP-II at 3.1 angstrom resolution. The structure revealed a 1:1 stoichiometry and a more extensive binding interface than anticipated from the paradigmatic two-site model. The structure helped rationalize a large body of mutagenesis data and together with modeling provided insights into CXCR4 interactions with its endogenous ligand CXCL12, its ability to recognize diverse ligands, and the specificity of CC and CXC receptors for their respective chemokines.

In mammals succinic semialdehyde dehydrogenase (SSADH) plays an essential role in the metabolism of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) to succinic acid (SA). Deficiency of SSADH in humans results in elevated levels of GABA and gamma-Hydroxybutyric acid (GHB), which leads to psychomotor retardation, muscular hypotonia, non-progressive ataxia and seizures. In Escherichia coli, two genetically distinct forms of SSADHs had been described that are essential for preventing accumulation of toxic levels of succinic semialdehyde (SSA) in cells. Here we structurally characterise SSADH encoded by the E coli gabD gene by X-ray crystallographic studies and compare these data with the structure of human SSADH. In the E. coli SSADH structure, electron density for the complete NADP+ cofactor in the binding sites is clearly evident; these data in particular revealing how the nicotinamide ring of the cofactor is positioned in each active site. Our structural data suggest that a deletion of three amino acids in E. coli SSADH permits this enzyme to use NADP+, whereas in contrast the human enzyme utilises NAD+. Furthermore, the structure of E. coli SSADH gives additional insight into human mutations that result in disease.

Opioids represent widely prescribed and abused medications, although their signal transduction mechanisms are not well understood. Here we present the 1.8 Å high-resolution crystal structure of the human δ-opioid receptor (δ-OR), revealing the presence and fundamental role of a sodium ion in mediating allosteric control of receptor functional selectivity and constitutive activity. The distinctive δ-OR sodium ion site architecture is centrally located in a polar interaction network in the seven-transmembrane bundle core, with the sodium ion stabilizing a reduced agonist affinity state, and thereby modulating signal transduction. Site-directed mutagenesis and functional studies reveal that changing the allosteric sodium site residue Asn 131 to an alanine or a valine augments constitutive β-arrestin-mediated signalling. Asp95Ala, Asn310Ala and Asn314Ala mutations transform classical δ-opioid antagonists such as naltrindole into potent β-arrestin-biased agonists. The data establish the molecular basis for allosteric sodium ion control in opioid signalling, revealing that sodium-coordinating residues act as ‘efficacy switches’ at a prototypic G-protein-coupled receptor. The 1.8 Å high-resolution X-ray crystal structure of the human δ-opioid receptor is presented, with site-directed mutagenesis and functional studies revealing a crucial role for a sodium ion in mediating allosteric control in this receptor. Making dual-action opioids Opioid receptors mediate the actions of endogenous and exogenous opioids for many physiological processes, including analgesia, consciousness, motor control and mood. This paper reports the X-ray crystal structure of the human δ-opioid receptor at 1.8 Å resolution, revealing the presence of a sodium ion that seems to mediate allosteric control of this G-protein-coupled receptor. Site-directed mutagenesis and functional studies show that mutating key amino acids in the allosteric sodium site to alanine transforms the antagonist naltrindole into a potent β-arrestin-biased agonist. Also apparent is an allosteric sodium-binding pocket that could aid the development of subtype-selective δ-opioid receptor agonists and antagonists — the extension of orthosteric ligands into the pocket could generate 'bitopic' orthosteric/allosteric compounds with more favourable pharmacological properties.

COOH-Terminal Clustering of Autoantibody and T-Cell Determinants on the Structure of GAD65 Provide Insights Into the Molecular Basis of Autoreactivity Gustavo Fenalti 1 , Christiane S. Hampe 2 , Yasir Arafat 1 , Ruby H.P. Law 1 , J. Paul Banga 3 , Ian R. Mackay 1 , James C. Whisstock 1 , Ashley M. Buckle 1 and Merrill J. Rowley 1 1 Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia 2 Department of Medicine, University of Washington, Seattle, Washington 3 School of Medicine of Kings College London, Division of Gene and Cell Based Therapy, Denmark Hill Campus, London, U.K Corresponding author: Prof. Merrill Rowley, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia. E-mail: merrill.rowley{at}med.monash.edu.au Abstract OBJECTIVE— To gain structural insights into the autoantigenic properties of GAD65 in type 1 diabetes, we analyzed experimental epitope mapping data in the context of the recently determined crystal structures of GAD65 and GAD67, to allow “molecular positioning” of epitope sites for B- and T-cell reactivity. RESEARCH DESIGN AND METHODS— Data were assembled from analysis of reported effects of mutagenesis of GAD65 on its reactivity with a panel of 11 human monoclonal antibodies (mAbs), supplemented by use of recombinant Fab to cross-inhibit reactivity with GAD65 by radioimmunoprecipitation of the same mAbs. RESULTS— The COOH-terminal region on GAD65 was the major autoantigenic site. B-cell epitopes were distributed within two separate clusters around different faces of the COOH-terminal domain. Inclusion of epitope sites in the pyridoxal phosphate–and NH 2 -terminal domains was attributed to the juxtaposition of all three domains in the crystal structure. Epitope preferences of different mAbs to GAD65 aligned with different clinical expressions of type 1 diabetes. Epitopes for four of five known reactive T-cell sequences restricted by HLA DRB1*0401 were aligned to solvent-exposed regions of the GAD65 structure and colocalized within the two B-cell epitope clusters. The continuous COOH-terminal epitope region of GAD65 was structurally highly flexible and therefore differed markedly from the equivalent region of GAD67. CONCLUSIONS— Structural features could explain the differing antigenicity, and perhaps immunogenicity, of GAD65 versus GAD67. The proximity of B- and T-cell epitopes within the GAD65 structure suggests that antigen-antibody complexes may influence antigen processing by accessory cells and thereby T-cell reactivity. GABA, γ-aminobutyric acid mAb, monoclonal antibody NHMRC, National Health and Medical Research Council PLP, pyridoxal phosphate rFab, recombinant Fab RIP, radioimmunoprecipitation Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on January 2008. DOI: 10.2337/db07-1461. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted January 4, 2008. Received October 12, 2007. DIABETES

The X-ray crystal structure of the human P2Y 12 receptor, which regulates platelet activation and thrombus formation, is solved in complex with an antithrombotic drug, providing insights for the development of new drugs. Key platelet aggregation GPCR structures Two papers in this issue of Nature present the crystal structures of the human P2Y 12 receptor, first in complex with the antithrombotic drug AZD1283, and second, bound to a full agonist (a close analogue of endogenous agonist ADP) and to a partial agonist. P2Y receptors are a family of purinergic G-protein-coupled receptors (GPCRs) that are activated by extracellular nucleotides. The P2Y 12 receptor is found mainly on the surface of platelets, where it regulates platelet activation and thrombus formation, and it is the target of several important antithrombotic drugs. In overall structure, P2Y 12 receptor is found to be similar to other GPCRs, although both the shape and location of the ligand-binding pocket are unusual. Comparisons of the three newly determined structures reveal that agonist binding induces a large-scale rearrangement of the extracellular domains of the GPCR. P2Y receptors (P2YRs), a family of purinergic G-protein-coupled receptors (GPCRs), are activated by extracellular nucleotides. There are a total of eight distinct functional P2YRs expressed in human, which are subdivided into P2Y 1 -like receptors and P2Y 12 -like receptors 1 . Their ligands are generally charged molecules with relatively low bioavailability and stability in vivo 2 , which limits our understanding of this receptor family. P2Y 12 R regulates platelet activation and thrombus formation 3 , 4 , and several antithrombotic drugs targeting P2Y 12 R—including the prodrugs clopidogrel (Plavix) and prasugrel (Effient) that are metabolized and bind covalently, and the nucleoside analogue ticagrelor (Brilinta) that acts directly on the receptor—have been approved for the prevention of stroke and myocardial infarction. However, limitations of these drugs (for example, a very long half-life of clopidogrel action and a characteristic adverse effect profile of ticagrelor) 5 , 6 suggest that there is an unfulfilled medical need for developing a new generation of P2Y 12 R inhibitors 7 , 8 . Here we report the 2.6 Å resolution crystal structure of human P2Y 12 R in complex with a non-nucleotide reversible antagonist, AZD1283. The structure reveals a distinct straight conformation of helix V, which sets P2Y 12 R apart from all other known class A GPCR structures. With AZD1283 bound, the highly conserved disulphide bridge in GPCRs between helix III and extracellular loop 2 is not observed and appears to be dynamic. Along with the details of the AZD1283-binding site, analysis of the extracellular interface reveals an adjacent ligand-binding region and suggests that both pockets could be required for dinucleotide binding. The structure provides essential insights for the development of improved P2Y 12 R ligands and allosteric modulators as drug candidates.

Chemokine receptors are critical regulators of cell migration in the context of immune surveillance, inflammation, and development. The G protein-coupled chemokine receptor CXCR4 is specifically implicated in cancer metastasis and HIV-1 infection. Here we report five independent crystal structures of CXCR4 bound to an antagonist small molecule IT1t and a cyclic peptide CVX15 at 2.5 to 3.2 angstrom resolution. All structures reveal a consistent homodimer with an interface including helices V and VI that may be involved in regulating signaling. The location and shape of the ligand-binding sites differ from other G protein-coupled receptors and are closer to the extracellular surface. These structures provide new clues about the interactions between CXCR4 and its natural ligand CXCL12, and with the HIV-1 glycoprotein gp120.