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Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/excretion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein, we report a new method termed “fixation-driven chemical cross-linking of exogenous ligands (FixEL), ” which traps and images exogenously administered molecules of interest (MOIs) in complex tissues. This method relies on protein-MOI interactions and chemical cross-linking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOIs such as the scaffold of PET tracer derivatives or drug-like small molecules., ホルマリン漬けから着想した小分子可視化法 --医薬品開発効率化につながる新たな戦略--. 京都大学プレスリリース. 2022-12-05.

Type-A γ-aminobutyric acid receptors (GABA(A)Rs) are pentameric ligand-gated chloride channels that mediate fast inhibitory signalling in neural circuits( 1,2 ) and are modulated by essential medicines including general anaesthetics and benzodiazepines( 3 ). Human GABA(A)R subunits are encoded by 19 paralogous genes which can, in theory, give rise to 495,235 receptor types. Yet, the principles governing the formation of pentamers, the permutational landscape of receptors that may emerge from a subunit set and the impact this has on GABA-ergic signalling remain largely unknown. Here, we use cryogenic electron microscopy (cryo-EM) to determine structures of extrasynaptic GABA(A)R assembled from α4, β3 and δ subunits, and their counterparts incorporating γ2 instead of δ subunits. In each case we could identify two receptor subtypes with distinct stoichiometries and arrangements, all four differing from those previously observed for synaptic, α1-containing receptors( 4–7 ). This, in turn, affects receptor responses to physiological and synthetic modulators by creating or eliminating ligand binding sites at subunit interfaces. We provide structural and functional evidence that selected GABA(A)R arrangements can act as coincidence detectors, simultaneously responding to two neurotransmitters, GABA and histamine. Using assembly simulations and single-cell RNA sequencing data( 8,9 ), we calculate upper bounds for receptor diversity in recombinant systems and in vivo. We propose that differential assembly is a pervasive mechanism for regulating the physiology and pharmacology of GABA(A)Rs.

The ability to incorporate a desired functionality into proteins of interest in a site-specific manner can provide powerful tools for investigating biological systems and creating therapeutic conjugates. However, there are not any universal methods that can be applied to all proteins, and it is thus important to explore the chemical strategy for protein modification. In this paper, we developed a new reactive peptide tag/probe pair system for site-specific covalent protein labeling. This method relies on the recognition-driven reaction of a peptide tag and a molecular probe, which comprises the lysine-containing short histidine tag (KH6 or H6K) and a binuclear nickel (II)- nitrilotriacetic acid (Ni2+-NTA) complex probe containing a lysine-reactive N-acyl-N-alkyl sulfonamide (NASA) group. The selective interaction of the His-tag and Ni2+-NTA propeles a rapid nucleophilic reaction between a lysine residue of the tag and the electrophilic NASA group of the probe by the proximity effect, resulting in the tag-site-specific functionalization of proteins. We characterized the reactive profile and site-specificity of this method using model peptides and proteins in vitro, and demonstrated the general utility for production of a nanobody-chemical probe conjugate without compromising its binding ability.

Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.

Synthetic excitatory synaptic organizer The human brain contains trillions of synapses within a vast network of neurons. Synapse remodeling is essential to ensure the efficient reception and integration of external stimuli and to store and retrieve information. Building and remodeling of synapses occurs throughout life under the control of synaptic organizer proteins. Errors in this process can lead to neuropsychiatric or neurological disorders. Suzuki et al. combined structural elements of natural synaptic organizers to develop an artificial version called CPTX, which has different binding properties (see the Perspective by Salinas). CPTX could act as a molecular bridge to reconnect neurons and restore excitatory synaptic function in animal models of cerebellar ataxia, familial Alzheimer's disease, and spinal cord injury. The findings illustrate how structure-guided approaches can help to repair neuronal circuits. Science , this issue p. eabb4853 ; see also p. 1052

Type-A γ-aminobutyric (GABAA) receptors are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and are common substances of abuse. Without reliable structural data, the mechanistic basis for the pharmacological modulation of GABAA receptors remains largely unknown. Here we report several high-resolution cryo-electron microscopy structures in which the full-length human α1β3γ2L GABAA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam. We describe the binding modes and mechanistic effects of these ligands, the closed and desensitized states of the GABAA receptor gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding region and the transmembrane, pore-forming region. This work provides a structural framework in which to integrate previous physiology and pharmacology research and a rational basis for the development of GABAA receptor modulators.

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.

14 páginas, 6 figuras, 1 tabla -- PAGS nros. 848-861, 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, This work was funded by Cancer Research UK, the Wellcome Trust, the UK Medical Research Council, the Spanish Ministry of Science and Innovation (SAF2008-00451, SAF2011-22988), the Red Temática de Investigación Cooperativa en Cáncer (RD06/0020/1001), Children's Hospital Boston Intellectual and Developmental Disabilities Research Center/National Institute of Child Health and Human Development (P30 HD-18655), and National Institute of General Medical Sciences (RO1 GM074241). S.C. is a recipient of the Chinese Ministry of Education-University of Oxford scholarship; D.B. is supported by EMBO; A.R.A. is a UK Medical Research Council Career Development Award Fellow; C.S. is a Wellcome Trust Research Career Development Fellow; X.H. is a Leukemia and Lymphoma Society Scholar; and E.Y.J. is a Cancer Research UK Principal Research Fellow

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.

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. © 2011 Elsevier Inc.

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.

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.

The complex pattern of neural connectivity established during nervous system development relies on the ability of the axon's motile tip, the growth cone, to receive, transduce, and integrate multiple environmental signals. Protein phosphorylation on tyrosine residues plays a key role in these processes (21, 29). Two major families of enzymes, the protein tyrosine kinases and the protein tyrosine phosphatases (PTPs), control cellular phosphotyrosine levels. These enzymes are found in both cytoplasmic and transmembrane (receptor-like) forms, and the biochemical interactions between them lead to a diversity of cellular behaviors (25, 76). Receptor protein tyrosine phosphatases (RPTPs) have recently joined the list of molecules involved in neural development and in particular in axon growth and guidance (reviewed in references 6, 68, 72, and 79). Type 2 RPTPs, containing cell adhesion molecule (CAM)-like extracellular regions, may be particularly well equipped to trigger signals involving cell-cell or cell-extracellular matrix contacts (68). Recent experiments with Drosophila have demonstrated the involvement of the RPTPs DLAR and DPTP69D in motor (19, 20, 50), retinal (27, 57), and midline (73) axon guidance. In leech, a LAR gene-related RPTP (HmLAR2) is implicated in Comb cell behavior, specifically in process outgrowth and mutual avoidance by sibling growth cones (2, 28). Several vertebrate RPTPs have been shown to promote neurite outgrowth in cell culture, including cPTPσ (51), RPTPκ (23), RPTPμ (10), and RPTPδ (82). Moreover, it has recently been shown that RPTPδ also has a potential guidance function, at least in vitro (74). In mice, gene deficiencies in type 2 RPTPs lead to various abnormalities. LAR deficiency leads to a reduction in size of basal forebrain cholinergic neurons, diminished hippocampal innervation, and defects in other tissues, such as the mammary gland (63, 78, 86). RPTPδ deficiency leads to impaired learning and enhanced hippocampal long-term potentiation (77). The most extreme defects are seen in RPTPσ-deficient mice, which show poor fecundity, hypomyelination of peripheral nerves, ataxias, and abnormalities in development of the hypothalamus and pituitary (24, 80). Although the developmental mechanism of these defects is not known, it is of interest that the avian orthologue of RPTPσ, cPTPσ (69, 85), regulates axon outgrowth of embryonic neurons (51). Despite this accumulation of functional data, much less is known about the extracellular cues that trigger signal transduction though RPTPs. Several RPTPs interact homophilically in trans, including RPTPμ (8), RPTPκ (62), and RPTPδ (82), but the effects of such homophilic interactions on enzyme function are as yet unclear. The laminin-nidogen complex has been shown to be a heterotypic ligand for a nonneural isoform of LAR (59), while type 5 RPTPζ can bind to several molecules, including contactin and tenascin and the cytokines midkine and pleiotrophin (60). Significantly, pleiotrophin has also recently been shown to suppress the catalytic activity of RPTPζ (53). cPTPσ, originally described as CRYPα (69), is a type 2 RPTP expressed as two major isoforms: cPTPσ1 (CRYPα1) has three immunoglobulin-like (Ig) domains and four fibronectin type III (FNIII) domains in its extracellular region, while cPTPσ2 (CRYPα2) has four extra FNIII domains. Both isoforms are strongly expressed in the chicken embryo nervous system, in particular within retinal and tectal axons and on their growth cones (70, 71). Moreover cPTPσ1 promotes intraretinal axon growth in vitro and controls growth cone morphology via the maintenance of lamellipodia (51). One or more ligands for cPTPσ are localized in the retinal and tectal basal lamina (BL) and on the glial endfeet of these tissues (33, 51), but the identity of these ligands has remained elusive. To understand how cPTPσ may function at the molecular and cellular levels, we sought to identify the molecular nature of these ligands. Here we show that cPTPσ is a heparin binding protein and that extracellular matrix heparan sulfate proteoglycans (HSPGs), in particular agrin and collagen XVIII, are binding partners for cPTPσ in vitro. Receptor affinity probe assays on tissue sections reveal that the binding of cPTPσ ectodomains to HSPGs is absolutely dependent on the presence of heparan sulfate (HS) side chains. Site-directed mutagenesis in a putative heparin and HS (heparin/HS) binding site in cPTPσ completely abolished this interaction. These data, together with the overlapping expression patterns of cPTPσ, agrin, and collagen XVIII in the developing chick retina, suggest that the reported interactions are physiologically relevant and that HSPGs could be a major ligand class for cPTPσ.

Correct functioning of the brain relies upon the precise connectivity between the billions of neurons that make up this crucial organ. Aberrations in the formation of these elaborate neural networks lead to neurodegenerative and neuropsychiatric disorders. A synapse-spanning molecular triad, involving members of the Neurexin, Cbln and ionotropic glutamate receptor delta families of proteins, is crucial for the accurate formation and proper function of synapses in the cerebellum. This trans-synaptic complex has been implicated in the molecular mechanisms behind motor control and motor learning, and furthermore individual members have been linked to diseases such as Alzheimer’s, autism spectrum disorders and schizophrenia. The major findings presented in this thesis include: crystal structures of the amino-terminal domains (ATD) of the two members of the ionotropic glutamate receptor delta (iGluR-Delta) family, functional characterisation of the effects of disrupting the ATD interface in one member of the iGluR-Delta family, a crystal structure of the C1q domain of Cbln1, biophysical analysis of the molecular interactions within the Neurexin-Cbln1-GluD2 trans-synaptic complex, as well as evidence for the domain arrangement of the ecto-domain of the iGluR-Delta proteins. Together, these data enhance our knowledge of the molecular details of this macro-molecular complex and provide evidence to support models for the mechanisms of their involvement in synapse formation and function, thereby making a contribution to the vast and medically relevant field of molecular neurobiology.

The receptor-like protein tyrosine phosphatase (RPTP) PTPσ controls the growth and targeting of retinal axons, both in culture and in ovo. Although the principal actions of PTPσ have been thought to be cell-autonomous, the possibility that RPTPs related to PTPσ 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 PTPσ ectodomains supports retinal neurite outgrowth in cell culture. We show that a receptor for PTPσ must exist on retinal axons and that binding of PTPσ to this receptor does not require the known, heparin binding properties of PTPσ. The neurite-promoting potential of PTPσ ectodomains requires a basic amino acid domain, previously demonstrated in vitro as being necessary for ligand binding by PTPσ. Furthermore, we demonstrate that heparin and oligosaccharide derivatives as short as 8mers, can specifically block neurite outgrowth on the PTPσ substrate, by competing for binding to this same domain. This is the first direct evidence of a non-cell-autonomous, neurite-promoting function of PTPσ and of a potential role for heparin-related oligosaccharides in modulating neurite promotion by an RPTP. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005

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.

Summary 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 β 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.

We review here recent results on the structure and function of a receptor protein tyrosine phosphatase, RPTPμ. 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 RPTPμ, the extracellular region is known to mediate homophilic adhesion. Sequence analysis indicates that it comprises six domains: an N-terminal MAM (meprin/A5/μ), one immunoglobulin-like domain and four fibronectin type III (FN) repeats. We have determined the crystal structure of the entire extracellular region for RPTPμ 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. © 2008 Biochemical Society.

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.

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., Receptor protein tyrosine phosphatase sigma (RPTPσ) promotes both neurite outgrowth and synaptic organization. Here, Coles et al. present the structural basis for this switch in function, whereby TrkC on the postsynaptic membrane and heparan sulphate proteoglycans compete for the same binding surface on RPTPσ.

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.

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.

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.

RGM Proteins Members of the repulsive guidance molecule (RGM) family of proteins can be secreted or reside on the surface of cells where they bind to the cell surface receptor, neogenin. The RGM proteins are named for their role in axon guidance for developing neurons, but their function is also linked to a range of human diseases, including inflammation, multiple sclerosis, and cancer. Bell et al. (p. 77 ) solved the crystal structures of the external portions of the RGMB protein with portions of neogenin. The structures revealed interactions of dimers of RGMB with neogenin in which ligand binding induced conformational changes that may initiate intracellular signaling from the receptor. RGM proteins contain a site of autocatalytic cleavage that affects secretion of the proteins, and some disease-associated mutations in RGM proteins were clustered at this site.

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.

Cell-cell contacts are fundamental to multicellular organisms and are subject to exquisite levels of control. Human RPTPμ 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 RPTPμ 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 RPTPμ ectodomain acts as a distance gauge and plays a key regulatory function, locking the phosphatase to its appropriate functional location.

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/TGFβ 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.

Nanobodies (Nbs) are popular and versatile tools for structural biology because they have a compact single immunoglobulin domain organization. Nbs bind their target proteins with high affinities while reducing their conformational heterogeneity, and they stabilize multi-protein complexes. Here we demonstrate that engineered Nbs can also help overcome two major obstacles that limit the resolution of single-particle cryo-EM reconstructions: particle size and preferential orientation at the water-air interface. We have developed and characterised novel constructs, termed megabodies, by grafting Nbs into selected protein scaffolds to increase their molecular weight while retaining the full antigen binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we used a megabody to solve the 2.5 Å resolution cryo-EM structure of a membrane protein that suffers from severe preferential orientation, the human GABAAβ3 homopentameric receptor bound to its small-molecule agonist histamine.

Summary During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a “trimer-of-trimers” super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs., Graphical abstract, Highlights • The NEO1-NET1 structure suggests NET1-mediated NEO1/DCC cell surface clustering • The receptor NEO1 and its ligands NET1 and RGM form a trimer-of-trimers supercomplex • NET1 inhibits RGMA-induced growth cone collapse via the NEO1-NET1-RGM complex • The NEO1-NET1-RGM complex silences RGM and NET1 effects on neuron migration, When extracellular guidance molecules Netrin and RGM that have opposing functions simultaneously bind the Neogenin receptor, a super-complex is formed that diminishes their functional outputs.