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In this issue of Structure, Fan et al. 1 report the structure of the full contactin 2 ectodomain, representing the first for the contactin family. The work reveals six immunoglobulin domains are essential for intercellular interactions, explores differences in proposed contactin 2 homodimerization mechanisms, and provides an updated model for contactin 2 organization on and between cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cartilage acidic protein-1 (CRTAC1) is a secreted glycoprotein with roles in development, function and repair of the nervous system. It is linked to ischemic stroke, osteoarthritis and (long) COVID outcomes, and has suppressive activity in carcinoma and bladder cancer. Structural characterization of CRTAC1 has been complicated by its tendency to form disulfide-linked aggregates. Here, we show that CRTAC1 is stabilized by potassium ions. Using x-ray crystallography, we determined the structure of CRTAC1 to 1.6 Å. This reveals that the protein consists of a three-domain fold, including a previously-unreported compact β-propeller-TTR combination, in which an extended loop of the TTR plugs the β-propeller core. Electron density is observed for ten bound ions: six calcium, three potassium and one sodium. Low potassium ion concentrations lead to changes in tryptophan environment and exposure of two buried free cysteines located on a β-blade and in the β-propeller-plugging TTR loop. Mutating the two free cysteines to serines prevents covalent intermolecular interactions, but not aggregation, in absence of potassium ions. The potassium ion binding sites are located between the blades of the β-propeller, explaining their importance for the stability of the CRTAC1 fold. Despite varying in sequence, the three potassium ion binding sites are structurally similar and conserved features of the CRTAC protein family. These insights into the stability and structure of CRTAC1 provide a basis for further work into the function of CRTAC1 in health and disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The FLAG-tag/anti-FLAG system is a widely used biochemical tool for protein detection and purification. Anti-FLAG M2 is the most popular antibody against the FLAG-tag, due to its ease of use, versatility, and availability in pure form or as bead conjugate. M2 binds N-terminal, C-terminal and internal FLAG-tags and binding is calcium-independent, but the molecular basis for the FLAG-tag specificity and recognition remains unresolved. Here we present an atomic resolution (1.17 Å) structure of the FLAG peptide in complex with the Fab of anti-FLAG M2, revealing key binding determinants. Five of the eight FLAG peptide residues form direct interactions with paratope residues. The FLAG peptide adopts a 3 10 helix conformation in complex with the Fab. These structural insights allowed us to rationally introduce point mutations on both the peptide and antibody side. We tested these by surface plasmon resonance, leading us to propose a shorter yet equally binding version of the FLAG-tag for the M2 antibody., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ‘Joost Snijder reports financial support was provided by Dutch Research Council. Bert Janssen reports financial support was provided by Dutch Research Council. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.’., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Neuronal network formation is facilitated by recognition between synaptic cell adhesion molecules at the cell surface. Alternative splicing of cell adhesion molecules provides additional specificity in forming neuronal connections. For the teneurin family of cell adhesion molecules, alternative splicing of the EGF-repeats and NHL domain controls synaptic protein-protein interactions. Here we present cryo-EM structures of the compact dimeric ectodomain of two teneurin-3 isoforms that harbour the splice insert in the EGF-repeats. This dimer is stabilised by an EGF8-ABD contact between subunits. Cryo-EM reconstructions of all four splice variants, together with SAXS and negative stain EM, reveal compacted dimers for each, with variant-specific dimeric arrangements. This results in specific trans-cellular interactions, as tested in cell clustering and stripe assays. The compact conformations provide a structural basis for teneurin homo- and heterophilic interactions. Altogether, our findings demonstrate how alternative splicing results in rearrangements of the dimeric subunits, influencing neuronal recognition and likely circuit wiring., (© 2024. The Author(s).)

The cell-surface attached glycoprotein contactin 2 is ubiquitously expressed in the nervous system and mediates homotypic cell-cell interactions to organize cell guidance, differentiation, and adhesion. Contactin 2 consists of six Ig and four fibronectin type III domains (FnIII) of which the first four Ig domains form a horseshoe structure important for homodimerization and oligomerization. Here we report the crystal structure of the six-domain contactin 2 Ig1-6 and show that the Ig5-Ig6 combination is oriented away from the horseshoe with flexion in interdomain connections. Two distinct dimer states, through Ig1-Ig2 and Ig3-Ig6 interactions, together allow formation of larger oligomers. Combined size exclusion chromatography with multiangle light scattering (SEC-MALS), small-angle X-ray scattering (SAXS) and native MS analysis indicates contactin 2 Ig1-6 oligomerizes in a glycan dependent manner. SAXS and negative-stain electron microscopy reveals inherent plasticity of the contactin 2 full-ectodomain. The combination of intermolecular binding sites and ectodomain plasticity explains how contactin 2 can function as a homotypic adhesion molecule in diverse intercellular environments., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cell-surface expressed contactin 1 and neurofascin 155 control wiring of the nervous system and interact across cells to form and maintain paranodal myelin-axon junctions. The molecular mechanism of contactin 1 - neurofascin 155 adhesion complex formation is unresolved. Crystallographic structures of complexed and individual contactin 1 and neurofascin 155 binding regions presented here, provide a rich picture of how competing and complementary interfaces, post-translational glycosylation, splice differences and structural plasticity enable formation of diverse adhesion sites. Structural, biophysical, and cell-clustering analysis reveal how conserved Ig1-2 interfaces form competing heterophilic contactin 1 - neurofascin 155 and homophilic neurofascin 155 complexes whereas contactin 1 forms low-affinity clusters through interfaces on Ig3-6. The structures explain how the heterophilic Ig1-Ig4 horseshoe's in the contactin 1 - neurofascin 155 complex define the 7.4 nm paranodal spacing and how the remaining six domains enable bridging of distinct intercellular distances., (© 2022. The Author(s).)

Establishment of correct synaptic connections is a crucial step during neural circuitry formation. The Teneurin family of neuronal transmembrane proteins promotes cell-cell adhesion via homophilic and heterophilic interactions, and is required for synaptic partner matching in the visual and hippocampal systems in vertebrates. It remains unclear how individual Teneurins form macromolecular cis- and trans-synaptic protein complexes. Here, we present a 2.7 Å cryo-EM structure of the dimeric ectodomain of human Teneurin4. The structure reveals a compact conformation of the dimer, stabilized by interactions mediated by the C-rich, YD-shell, and ABD domains. A 1.5 Å crystal structure of the C-rich domain shows three conserved calcium binding sites, and thermal unfolding assays and SAXS-based rigid-body modeling demonstrate that the compactness and stability of Teneurin4 dimers are calcium-dependent. Teneurin4 dimers form a more extended conformation in conditions that lack calcium. Cellular assays reveal that the compact cis-dimer is compatible with homomeric trans-interactions. Together, these findings support a role for teneurins as a scaffold for macromolecular complex assembly and the establishment of cis- and trans-synaptic interactions to construct functional neuronal circuits., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: The epidermal growth factor receptor (EGFR) is involved in various developmental processes, and alterations of its extracellular segment are associated with several types of cancers, in particular glioblastoma multiforme (GBM). The EGFR extracellular region is therefore a primary target for therapeutic agents, such as monoclonal antibodies and variable domains of heavy chain antibodies (VHH), also called nanobodies. Nanobodies have been previously shown to bind to EGFR, and to inhibit ligand-mediated EGFR activation., Results: Here we present the X-ray crystal structures of the EgB4 nanobody, alone (to 1.48 Å resolution) and bound to the full extracellular EGFR-EGF complex in its active conformation (to 6.0 Å resolution). We show that EgB4 binds to a new epitope located on EGFR domains I and II, and we describe the molecular mechanism by which EgB4 plays a non-inhibitory role in EGFR signaling., Conclusion: This work provides the structural basis for the application of EgB4 as a tool for research, for targeted therapy, or as a biomarker to locate EGFR-associated tumors, all without affecting EGFR activation., (© 2022. The Author(s).)

The continued rise of antibiotic resistance is a global concern that threatens to undermine many aspects of modern medical practice. Key to addressing this threat is the discovery and development of new antibiotics that operate by unexploited modes of action. The so-called calcium-dependent lipopeptide antibiotics (CDAs) are an important emerging class of natural products that provides a source of new antibiotic agents rich in structural and mechanistic diversity. Notable in this regard is the subset of CDAs comprising the laspartomycins and amphomycins/friulimicins that specifically target the bacterial cell wall precursor undecaprenyl phosphate (C 55 -P). In this study we describe the design and synthesis of new C 55 -P-targeting CDAs with structural features drawn from both the laspartomycin and amphomycin/friulimicin classes. Assessment of these lipopeptides revealed previously unknown and surprisingly subtle structural features that are required for antibacterial activity. High-resolution crystal structures further indicate that the amphomycin/friulimicin-like lipopeptides adopt a unique crystal packing that governs their interaction with C 55 -P and provides an explanation for their antibacterial effect. In addition, live-cell microscopy studies provide further insights into the biological activity of the C 55 -P targeting CDAs highlighting their unique mechanism of action relative to the clinically used CDA daptomycin., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The aim of this study was to get insight into the internalization and transport of PEGylat-ed mixed micelles loaded by vitamin K, as mediated by Scavenger Receptor B1 (SR-B1) that is abundantly expressed by intestinal epithelium cells as well as by differentiated Caco-2 cells. Inhibition of SR-B1 reduced endocytosis and transport of vitamin-K-loaded 0%, 30% and 50% PEGylated mixed micelles and decreased colocalization of the micelles with SR-B1. Confocal fluorescence microscopy, fluorescence-activated cell sorting (FACS) analysis, and surface plasmon resonance (SPR) were used to study the interaction between the mixed micelles of different compositions (varying vitamin K loading and PEG content) and SR-B1. Interaction of PEGylated micelles was independent of the vitamin K content, indicating that the PEG shell prevented vitamin K exposure at the surface of the micelles and binding with the receptor and that the PEG took over the micelles' ability to bind to the receptor. Molecular docking calculations corroborated the dual binding of both vita-min K and PEG with the binding domain of SR-B1. In conclusion, the improved colloidal stability of PEGylated mixed micelles did not compromise their cellular uptake and transport due to the affinity of PEG for SR-B1. SR-B1 is able to interact with PEGylated nanoparticles and mediates their subsequent internalization and transport.

The Notch signaling system links cellular fate to that of its neighbors, driving proliferation, apoptosis, and cell differentiation in metazoans, whereas dysfunction leads to debilitating developmental disorders and cancers. Other than a five-by-five domain complex, it is unclear how the 40 extracellular domains of the Notch1 receptor collectively engage the 19 domains of its canonical ligand, Jagged1, to activate Notch1 signaling. Here, using cross-linking mass spectrometry (XL-MS), biophysical, and structural techniques on the full extracellular complex and targeted sites, we identify five distinct regions, two on Notch1 and three on Jagged1, that form an interaction network. The Notch1 membrane-proximal regulatory region individually binds to the established Notch1 epidermal growth factor (EGF) 8-EGF13 and Jagged1 C2-EGF3 activation sites as well as to two additional Jagged1 regions, EGF8-EGF11 and cysteine-rich domain. XL-MS and quantitative interaction experiments show that the three Notch1-binding sites on Jagged1 also engage intramolecularly. These interactions, together with Notch1 and Jagged1 ectodomain dimensions and flexibility, determined by small-angle X-ray scattering, support the formation of nonlinear architectures. Combined, the data suggest that critical Notch1 and Jagged1 regions are not distal but engage directly to control Notch1 signaling, thereby redefining the Notch1-Jagged1 activation mechanism and indicating routes for therapeutic applications., Competing Interests: The authors declare no competing interest.

Ticks, as blood-sucking parasites, have developed a complex strategy to evade and suppress host immune responses during feeding. The crucial part of this strategy is expression of a broad family of salivary proteins, called Evasins, to neutralize chemokines responsible for cell trafficking and recruitment. However, structural information about Evasins is still scarce, and little is known about the structural determinants of their binding mechanism to chemokines. Here, we studied the structurally uncharacterized Evasin-4, which neutralizes a broad range of CC-motif chemokines, including the chemokine CC-motif ligand 5 (CCL5) involved in atherogenesis. Crystal structures of Evasin-4 and E66S CCL5, an obligatory dimeric variant of CCL5, were determined to a resolution of 1.3-1.8 Å. The Evasin-4 crystal structure revealed an L-shaped architecture formed by an N- and C-terminal subdomain consisting of eight β-strands and an α-helix that adopts a substantially different position compared with closely related Evasin-1. Further investigation into E66S CCL5-Evasin-4 complex formation with NMR spectroscopy showed that residues of the N terminus are involved in binding to CCL5. The peptide derived from the N-terminal region of Evasin-4 possessed nanomolar affinity to CCL5 and inhibited CCL5 activity in monocyte migration assays. This suggests that Evasin-4 derivatives could be used as a starting point for the development of anti-inflammatory drugs., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Denisov et al.)

Type-I transmembrane proteins represent a large group of 1,412 proteins in humans with a multitude of functions in cells and tissues. They are characterized by an extracellular, or luminal, N-terminus followed by a single transmembrane helix and a cytosolic C-terminus. The domain composition and structures of the extracellular and intercellular segments differ substantially amongst its members. Most of the type-I transmembrane proteins have roles in cell signaling processes, as ligands or receptors, and in cellular adhesion. The extracellular segment often determines specificity and can control signaling and adhesion. Here we focus on recent structural understanding on how the extracellular segments of several diverse type-I transmembrane proteins engage in interactions and can undergo conformational changes for their function. Interactions at the extracellular side by proteins on the same cell or between cells are enhanced by the transmembrane setting. Extracellular conformational domain rearrangement and structural changes within domains alter the properties of the proteins and are used to regulate signaling events. The combination of structural properties and interactions can support the formation of larger-order assemblies on the membrane surface that are important for cellular adhesion and intercellular signaling., (Copyright © 2020 Chataigner, Leloup and Janssen.)

Background: Olfactomedin-1 (Olfm1; also known as Noelin or Pancortin) is a highly-expressed secreted brain and retina protein and its four isoforms have different roles in nervous system development and function. Structural studies showed that the long Olfm1 isoform BMZ forms a disulfide-linked tetramer with a V-shaped architecture. The tips of the Olfm1 "V" each consist of two C-terminal β-propeller domains that enclose a calcium binding site. Functional characterisation of Olfm1 may be aided by new biochemical tools derived from these core structural elements., Results: Here we present the production, purification and structural analysis of three novel monomeric, dimeric and tetrameric forms of mammalian Olfm1 for functional studies. We characterise these constructs structurally by high-resolution X-ray crystallography and small-angle X-ray scattering. The crystal structure of the Olfm1 β-propeller domain (to 1.25 Å) represents the highest-resolution structure of an olfactomedin family member to date, revealing features such as a hydrophilic tunnel containing water molecules running into the core of the domain where the calcium binding site resides. The shorter Olfactomedin-1 isoform BMY is a disulfide-linked tetramer with a shape similar to the corresponding region in the longer BMZ isoform., Conclusions: These recombinantly-expressed protein tools should assist future studies, for example of biophysical, electrophysiological or morphological nature, to help elucidate the functions of Olfm1 in the mature mammalian brain. The control over the oligomeric state of Olfm1 provides a firm basis to better understand the role of Olfm1 in the (trans-synaptic) tethering or avidity-mediated clustering of synaptic receptors such as post-synaptic AMPA receptors and pre-synaptic amyloid precursor protein. In addition, the variation in domain composition of these protein tools provides a means to dissect the Olfm1 regions important for receptor binding.

Murine paired immunoglobulin receptor B (PirB) and its human ortholog leukocyte immunoglobulin-like receptor B2 (LILRB2) are widely expressed inhibitory receptors that interact with a diverse set of extracellular ligands and exert functions ranging from down-regulation of immune responses to inhibition of neuronal growth. However, structural information that could shed light on how PirB interacts with its ligands is lacking. Here, we report crystal structures of the PirB ectodomain; the first full ectodomain structure for a LILR family member, at 3.3-4.5 Å resolution. The structures reveal that PirB's six Ig-like domains are arranged at acute angles, similar to the structures of leukocyte immunoglobulin-like receptor (LILR) and killer-cell immunoglobulin-like receptor (KIR). We observe that this regular arrangement is followed throughout the ectodomain, resulting in an extended zigzag conformation. In two out of the five structures reported here, the repeating zigzag is broken by the first domain that can adopt two alternative orientations. Quantitative binding experiments revealed a 9 μm dissociation constant for PirB-myelin-associated glycoprotein (MAG) ectodomain interactions. Taken together, these structural findings and the observed PirB-MAG interactions are compatible with a model for intercellular signaling in which the PirB extracellular domains, which point away from the cell surface, enable interaction with ligands in trans ., (© 2019 Vlieg et al.)

Pioneering bioinformatic analysis using sequence data revealed that teneurins evolved from bacterial tyrosine-aspartate (YD)-repeat protein precursors. Here, we discuss how structures of the C-terminal domain of teneurins, determined using X -ray crystallography and electron microscopy, support the earlier findings on the proteins' ancestry. This chapter describes the structure of the teneurin scaffold with reference to a large family of teneurin-like proteins that are widespread in modern prokaryotes. The central scaffold of modern eukaryotic teneurins is decorated by additional domains typically found in bacteria, which are re-purposed in eukaryotes to generate highly multifunctional receptors. We discuss how alternative splicing contributed to further diversifying teneurin structure and thereby function. This chapter traces the evolution of teneurins from a structural point of view and presents the state-of-the-art of how teneurin function is encoded by its specific structural features.

Signaling of SorCS receptors by proneurotrophin ligands regulates neuronal plasticity, induces apoptosis and is associated with mental disorders. The detailed structure of SorCS2 and its extracellular specificity are unresolved. Here we report crystal structures of the SorCS2-NGF complex and unliganded SorCS2 ectodomain, revealing cross-braced SorCS2 homodimers with two NGF dimers bound in a 2:4 stoichiometry. Five out of six SorCS2 domains directly contribute to dimer formation and a C-terminal membrane proximal unreported domain, with an RNA recognition motif fold, locks the dimer in an intermolecular head-to-tail interaction. The complex structure shows an altered SorCS2 conformation indicating substantial structural plasticity. Both NGF dimer chains interact exclusively with the top face of a SorCS2 β-propeller. Biophysical experiments reveal that NGF, proNGF, and proBDNF bind at this site on SorCS2. Taken together, our data reveal a structurally flexible SorCS2 receptor that employs the large β-propeller as a ligand binding platform.

Teneurins are ancient cell-cell adhesion receptors that are vital for brain development and synapse organisation. They originated in early metazoan evolution through a horizontal gene transfer event when a bacterial YD-repeat toxin fused to a eukaryotic receptor. We present X-ray crystallography and cryo-EM structures of two Teneurins, revealing a ~200 kDa extracellular super-fold in which eight sub-domains form an intricate structure centred on a spiralling YD-repeat shell. An alternatively spliced loop, which is implicated in homophilic Teneurin interaction and specificity, is exposed and thus poised for interaction. The N-terminal side of the shell is 'plugged' via a fibronectin-plug domain combination, which defines a new class of YD proteins. Unexpectedly, we find that these proteins are widespread amongst modern bacteria, suggesting early metazoan receptor evolution from a distinct class of proteins, which today includes both bacterial proteins and eukaryotic Teneurins.

The calcium-dependent antibiotics (CDAs) are an important emerging class of antibiotics. The crystal structure of the CDA laspartomycin C in complex with calcium and the ligand geranyl-phosphate at a resolution of 1.28 Å is reported. This is the first crystal structure of a CDA bound to its bacterial target. The structure is also the first to be reported for an antibiotic that binds the essential bacterial phospholipid undecaprenyl phosphate (C 55 -P). These structural insights are of great value in the design of antibiotics capable of exploiting this unique bacterial target., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Low pH-induced ligand release and receptor recycling are important steps for endocytosis. The transmembrane protein sortilin, a β-propeller containing endocytosis receptor, internalizes a diverse set of ligands with roles in cell differentiation and homeostasis. The molecular mechanisms of pH-mediated ligand release and sortilin recycling are unresolved. Here we present crystal structures that show the sortilin luminal segment (s-sortilin) undergoes a conformational change and dimerizes at low pH. The conformational change, within all three sortilin luminal domains, provides an altered surface and the dimers sterically shield a large interface while bringing the two s-sortilin C-termini into close proximity. Biophysical and cell-based assays show that members of two different ligand families, (pro)neurotrophins and neurotensin, preferentially bind the sortilin monomer. This indicates that sortilin dimerization and conformational change discharges ligands and triggers recycling. More generally, this work may reveal a double mechanism for low pH-induced ligand release by endocytosis receptors.

The Nogo Receptor (NgR) is a glycophosphatidylinositol-anchored cell-surface protein and is a receptor for three myelin-associated inhibitors of regeneration: myelin-associated glycoprotein, Nogo66 and oligodendrocyte myelin glycoprotein. In combination with different co-receptors, NgR mediates signalling that reduces neuronal plasticity. The available structures of the NgR ligand-binding leucine-rich repeat (LRR) domain have an artificial disulfide pattern owing to truncated C-terminal construct boundaries. NgR has previously been shown to self-associate via its LRR domain, but the structural basis of this interaction remains elusive. Here, crystal structures of the NgR LRR with a longer C-terminal segment and a native disulfide pattern are presented. An additional C-terminal loop proximal to the C-terminal LRR cap is stabilized by two newly formed disulfide bonds, but is otherwise mostly unstructured in the absence of any stabilizing interactions. NgR crystallized in six unique crystal forms, three of which share a crystal-packing interface. NgR crystal-packing interfaces from all eight unique crystal forms are compared in order to explore how NgR could self-interact on the neuronal plasma membrane.

Myelin-associated glycoprotein (MAG) is a myelin-expressed cell-adhesion and bi-directional signalling molecule. MAG maintains the myelin-axon spacing by interacting with specific neuronal glycolipids (gangliosides), inhibits axon regeneration and controls myelin formation. The mechanisms underlying MAG adhesion and signalling are unresolved. We present crystal structures of the MAG full ectodomain, which reveal an extended conformation of five Ig domains and a homodimeric arrangement involving membrane-proximal domains Ig4 and Ig5. MAG-oligosaccharide complex structures and biophysical assays show how MAG engages axonal gangliosides at domain Ig1. Two post-translational modifications were identified-N-linked glycosylation at the dimerization interface and tryptophan C-mannosylation proximal to the ganglioside binding site-that appear to have regulatory functions. Structure-guided mutations and neurite outgrowth assays demonstrate MAG dimerization and carbohydrate recognition are essential for its regeneration-inhibiting properties. The combination of trans ganglioside binding and cis homodimerization explains how MAG maintains the myelin-axon spacing and provides a mechanism for MAG-mediated bi-directional signalling.

Surface plasmon resonance (SPR) is one of the most powerful label-free methods to determine the kinetic parameters of molecular interactions in real time and in a highly sensitive way. Penicillin-binding proteins (PBPs) are peptidoglycan synthesis enzymes present in most bacteria. Established protocols to analyze interactions of PBPs by SPR involve immobilization to an ampicillin-coated chip surface (a β-lactam antibiotic mimicking its substrate), thereby forming a covalent complex with the PBPs transpeptidase (TP) active site. However, PBP interactions measured with a substrate-bound TP domain potentially affect interactions near the TPase active site. Furthermore, in vivo PBPs are anchored in the inner membrane by an N-terminal transmembrane helix, and hence immobilization at the C-terminal TPase domain gives an orientation contrary to the in vivo situation. We designed a new procedure: immobilization of PBP by copper-free click chemistry at an azide incorporated in the N terminus. In a proof-of-principle study, we immobilized Escherichia coli PBP1B on an SPR chip surface and used this for the analysis of the well-characterized interaction of PBP1B with LpoB. The site-specific incorporation of the azide affords control over protein orientation, thereby resulting in a homogeneous immobilization on the chip surface. This method can be used to study topology-dependent interactions of any (membrane) protein., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Class A plexins (PlxnAs) act as semaphorin receptors and control diverse aspects of nervous system development and plasticity, ranging from axon guidance and neuron migration to synaptic organization. PlxnA signaling requires cytoplasmic domain dimerization, but extracellular regulation and activation mechanisms remain unclear. Here we present crystal structures of PlxnA (PlxnA1, PlxnA2, and PlxnA4) full ectodomains. Domains 1-9 form a ring-like conformation from which the C-terminal domain 10 points away. All our PlxnA ectodomain structures show autoinhibitory, intermolecular "head-to-stalk" (domain 1 to domain 4-5) interactions, which are confirmed by biophysical assays, live cell fluorescence microscopy, and cell-based and neuronal growth cone collapse assays. This work reveals a 2-fold role of the PlxnA ectodomains: imposing a pre-signaling autoinhibitory separation for the cytoplasmic domains via intermolecular head-to-stalk interactions and supporting dimerization-based PlxnA activation upon ligand binding. More generally, our data identify a novel molecular mechanism for preventing premature activation of axon guidance receptors., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Unlabelled: The A-kinase anchoring protein (AKAP) smAKAP has three extraordinary features; it is very small, it is anchored directly to membranes by acyl motifs, and it interacts almost exclusively with the type I regulatory subunits (RI) of cAMP-dependent kinase (PKA). Here, we determined the crystal structure of smAKAP's A-kinase binding domain (smAKAP-AKB) in complex with the dimerization/docking (D/D) domain of RIα which reveals an extended hydrophobic interface with unique interaction pockets that drive smAKAP's high specificity for RI subunits. We also identify a conserved PKA phosphorylation site at Ser66 in the AKB domain which we predict would cause steric clashes and disrupt binding. This correlates with in vivo colocalization and fluorescence polarization studies, where Ser66 AKB phosphorylation ablates RI binding. Hydrogen/deuterium exchange studies confirm that the AKB helix is accessible and dynamic. Furthermore, full-length smAKAP as well as the unbound AKB is predicted to contain a break at the phosphorylation site, and circular dichroism measurements confirm that the AKB domain loses its helicity following phosphorylation. As the active site of PKA's catalytic subunit does not accommodate α-helices, we predict that the inherent flexibility of the AKB domain enables its phosphorylation by PKA. This represents a novel mechanism, whereby activation of anchored PKA can terminate its binding to smAKAP affecting the regulation of localized cAMP signaling events., Database: Structural data are available in the PDB under accession number 5HVZ., (© 2016 Federation of European Biochemical Societies.)

Olfactomedin-1 (Olfm1; also known as noelin and pancortin) is a member of the olfactomedin domain-containing superfamily and a highly expressed neuronal glycoprotein important for nervous system development. It binds a number of secreted proteins and cell surface-bound receptors to induce cell signaling processes. Using a combined approach of x-ray crystallography, solution scattering, analytical ultracentrifugation, and electron microscopy we determined that full-length Olfm1 forms disulfide-linked tetramers with a distinctive V-shaped architecture. The base of the "V" is formed by two disulfide-linked dimeric N-terminal domains. Each of the two V legs consists of a parallel dimeric disulfide-linked coiled coil with a C-terminal β-propeller dimer at the tips. This agrees with our crystal structure of a C-terminal coiled-coil segment and β-propeller combination (Olfm1(coil-Olf)) that reveals a disulfide-linked dimeric arrangement with the β-propeller top faces in an outward exposed orientation. Similar to its family member myocilin, Olfm1 is stabilized by calcium. The dimer-of-dimers architecture suggests a role for Olfm1 in clustering receptors to regulate signaling and sheds light on the conformation of several other olfactomedin domain family members., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

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

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

Immune protection by the complement system critically depends on assembly of C3 convertases on the surface of pathogens and altered host cells. These short-lived protease complexes are formed through pro-convertases, which for the alternative pathway consist of the complement component C3b and the pro-enzyme factor B (FB). Here, we present the crystal structure at 2.2-A resolution, small-angle X-ray scattering and electron microscopy (EM) data of the pro-convertase formed by human FB and cobra venom factor (CVF), a potent homologue of C3b that generates more stable convertases. FB is loaded onto CVF through its pro-peptide Ba segment by specific contacts, which explain the specificity for the homologous C3b over the native C3 and inactive products iC3b and C3c. The protease segment Bb binds the carboxy terminus of CVF through the metal-ion dependent adhesion site of the Von Willebrand factor A-type domain. A possible dynamic equilibrium between a 'loading' and 'activation' state of the pro-convertase may explain the observed difference between the crystal structure of CVFB and the EM structure of C3bB. These insights into formation of convertases provide a basis for further development of complement therapeutics.

Activation of the complement system generates potent chemoattractants and leads to the opsonization of cells for immune clearance. Short-lived protease complexes cleave complement component C3 into anaphylatoxin C3a and opsonin C3b. Here we report the crystal structure of the C3 convertase formed by C3b and the protease fragment Bb, which was stabilized by the bacterial immune-evasion protein SCIN. The data suggest that the proteolytic specificity and activity depend on the formation of dimers of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of the complex of C3b and Bb is crucial to complement regulation and was determined by slow binding kinetics of the Mg(2+)-adhesion site in Bb. Understanding the mechanistic basis of the central complement-activation step and microbial immune evasion strategies targeting this step will aid in the development of complement therapeutics.

Factor H (FH) is an abundant regulator of complement activation and protects host cells from self-attack by complement. Here we provide insight into the regulatory activity of FH by solving the crystal structure of the first four domains of FH in complex with its target, complement fragment C3b. FH interacted with multiple domains of C3b, covering a large, extended surface area. The structure indicated that FH destabilizes the C3 convertase by competition and electrostatic repulsion and that FH enables proteolytic degradation of C3b by providing a binding platform for protease factor I while stabilizing the overall domain arrangement of C3b. Our results offer general models for complement regulation and provide structural explanations for disease-related mutations in the genes encoding both FH and C3b.

Cobra venom factor (CVF) is a structural and functional analog of complement C3 isolated from cobra venom. Both CVF and C3b can bind factor B and subsequently form the bimolecular C3/C5 convertases CVF,Bb or C3b,Bb, respectively. The two homologous enzymes exhibit several differences of which the difference in physico-chemical stability is most important, allowing continuous activation of C3 and C5 by CVF,Bb, leading to serum complement depletion. Here we describe the detailed functional properties of two hybrid proteins in which the 113 or 315 C-terminal residues of C3 were replaced with corresponding CVF sequences. Both hybrid proteins formed stable convertases that exhibited C3-cleaving activity, although at different rates. Neither convertase cleaved C5. Both convertases showed partial resistance to inactivation by factors H and I, allowing them to deplete complement in human serum. These data demonstrate that functionally important structural differences between CVF and C3 are located in the very C-terminal region of both homologous proteins, and that small substitutions in human C3 with homologous CVF sequence result in C3 derivatives with CVF-like functions. Such hybrid proteins are important tools to study the structure/function relationships in both C3 and CVF, and these "humanized CVF" proteins may become reagents for therapeutic complement depletion.

Atypical hemolytic uremic syndrome (aHUS) is a disease of complement dysregulation. In approximately 50% of patients, mutations have been described in the genes encoding the complement regulators factor H, MCP, and factor I or the activator factor B. We report here mutations in the central component of the complement cascade, C3, in association with aHUS. We describe 9 novel C3 mutations in 14 aHUS patients with a persistently low serum C3 level. We have demonstrated that 5 of these mutations are gain-of-function and 2 are inactivating. This establishes C3 as a susceptibility factor for aHUS.

Complement in mammalian plasma recognizes pathogenic, immunogenic and apoptotic cell surfaces, promotes inflammatory responses and marks particles for cell lysis, phagocytosis and B-cell stimulation. At the heart of the complement system are two large proteins, complement component C3 and protease factor B. These two proteins are pivotal for amplification of the complement response and for labelling of the target particles, steps that are required for effective clearance of the target. Here we review the molecular mechanisms of complement activation, in which proteolysis and complex formation result in large conformational changes that underlie the key offensive step of complement executed by C3 and factor B. Insights into the mechanisms of complement amplification are crucial for understanding host defence and pathogen immune evasion, and for the development of complement-immune therapies.

Undesired complement activation is a major cause of tissue injury in various pathological conditions and contributes to several immune complex diseases. Compstatin, a 13-residue peptide, is an effective inhibitor of the activation of complement component C3 and thus blocks a central and crucial step in the complement cascade. The precise binding site on C3, the structure in the bound form, and the exact mode of action of compstatin are unknown. Here we present the crystal structure of compstatin in complex with C3c, a major proteolytic fragment of C3. The structure reveals that the compstatin-binding site is formed by the macroglobulin (MG) domains 4 and 5. This binding site is part of the structurally stable MG-ring formed by domains MG 1-6 and is far away from any other known binding site on C3. Compstatin does not alter the conformation of C3c, whereas compstatin itself undergoes a large conformational change upon binding. We propose a model in which compstatin sterically hinders the access of the substrate C3 to the convertase complexes, thus blocking complement activation and amplification. These insights are instrumental for further development of compstatin as a potential therapeutic.

Activation of the protein C3 into C3b in the complement pathway is a crucial step in the complement immune response against pathogenic, immunogenic and apoptotic particles. Ajees et al. describe a crystal structure for C3b that deviates from the one reported by Janssen et al. and by Wiesmann et al.. We have reanalysed the data deposited by Ajees et al. and have discovered features that are inconsistent with the known physical properties of macromolecular structures and their diffraction data. Our findings therefore call into question the crystal structure for C3b reported by Ajees et al..

Factor B is the central protease of the complement system of immune defense. Here, we present the crystal structure of human factor B at 2.3-A resolution, which reveals how the five-domain proenzyme is kept securely inactive. The canonical activation helix of the Von Willebrand factor A (VWA) domain is displaced by a helix from the preceding domain linker. The two helices conformationally link the scissile-activation peptide and the metal ion-dependent adhesion site required for binding of the ligand C3b. The data suggest that C3b binding displaces the three N-terminal control domains and reshuffles the two central helices. Reshuffling of the helices releases the scissile bond for final proteolytic activation and generates a new interface between the VWA domain and the serine protease domain. This allosteric mechanism is crucial for tight regulation of the complement-amplification step in the immune response.

C3 is a central protein of the complement system, which is important to immune defense and provides a link between innate and adaptive immunity. Three pathways of complement activation converge at the activation of C3 yielding a diverse set of biological responses. This versatile and flexible molecule interacts with various proteins to fulfill its functions. Here we review recent insights gained from the crystal structure determinations of human, native C3 and its physiological down-regulation product C3c. The data provided, for the first time, a complete and detailed view of the composition and arrangement of the domains in C3. Comparison of C3 with C3c indicates marked flexibility of the molecule, particularly in the alpha-chain. We discuss the observed domain rearrangements, conformational changes and the location of various protein binding sites. These detailed, and structural, insights are important for developing models of the molecular mechanisms underlying the diverse biological activities of this large and complex molecule.

Resistance to infection and clearance of cell debris in mammals depend on the activation of the complement system, which is an important component of innate and adaptive immunity. Central to the complement system is the activated form of C3, called C3b, which attaches covalently to target surfaces to amplify complement response, label cells for phagocytosis and stimulate the adaptive immune response. C3b consists of 1,560 amino-acid residues and has 12 domains. It binds various proteins and receptors to effect its functions. However, it is not known how C3 changes its conformation into C3b and thereby exposes its many binding sites. Here we present the crystal structure at 4-A resolution of the activated complement protein C3b and describe the conformational rearrangements of the 12 domains that take place upon proteolytic activation. In the activated form the thioester is fully exposed for covalent attachment to target surfaces and is more than 85 A away from the buried site in native C3 (ref. 5). Marked domain rearrangements in the alpha-chain present an altered molecular surface, exposing hidden and cryptic sites that are consistent with known putative binding sites of factor B and several complement regulators. The structural data indicate that the large conformational changes in the proteolytic activation and regulation of C3 take place mainly in the first conversion step, from C3 to C3b. These insights are important for the development of strategies to treat immune disorders that involve complement-mediated inflammation.

Background: The complement system has a critical role in both the innate and the adaptive immune responses. In humans, C3 exists as two main allotypes, F (fast) and S (slow), which are known to affect the incidence of inflammatory disease. We conducted a study to address the influence of these alleles on late renal-graft outcome., Methods: We determined the C3 allotypes of 662 pairs of adult kidney donors and recipients from 1993 through 2002 and then related C3F/S polymorphism status to demographic and clinical outcome data. The median length of follow-up was 3.3 years., Results: Analysis of 513 pairs of white donors and recipients identified 113 C3S/S recipients of a C3S/F or a C3F/F kidney and 179 C3S/S recipients of a C3S/S kidney. Graft survival was significantly better with a C3F/F or C3F/S donor allotype than a C3S/S allotype (P=0.05). The hazard ratio for graft loss of C3S/S kidneys, as compared with C3F/F or C3F/S kidneys, was 2.21 (95 percent confidence interval, 1.04 to 4.72; P=0.04). The graft function of C3F/F or C3F/S donor kidneys was significantly better than that of C3S/S donor kidneys (P<0.001). The effect of the C3F allele was specific to recipients who did not themselves possess this allele. Multivariate analysis excluded effects of other factors known to influence graft outcome., Conclusions: Expression of C3 alleles by donor renal cells appears to have a differential effect on late graft outcome. Among white C3S/S recipients, receipt of a C3F/F or C3F/S donor kidney, rather than a C3S/S donor kidney, is associated with a significantly better long-term outcome. These findings suggest that the two alleles have functional differences., (Copyright 2006 Massachusetts Medical Society.)

The mammalian complement system is a phylogenetically ancient cascade system that has a major role in innate and adaptive immunity. Activation of component C3 (1,641 residues) is central to the three complement pathways and results in inflammation and elimination of self and non-self targets. Here we present crystal structures of native C3 and its final major proteolytic fragment C3c. The structures reveal thirteen domains, nine of which were unpredicted, and suggest that the proteins of the alpha2-macroglobulin family evolved from a core of eight homologous domains. A double mechanism prevents hydrolysis of the thioester group, essential for covalent attachment of activated C3 to target surfaces. Marked conformational changes in the alpha-chain, including movement of a critical interaction site through a ring formed by the domains of the beta-chain, indicate an unprecedented, conformation-dependent mechanism of activation, regulation and biological function of C3.

The structure of a novel plant defensin isolated from the flowers of Petunia hybrida has been determined by (1)H NMR spectroscopy. P. hybrida defensin 1 (PhD1) is a basic, cysteine-rich, antifungal protein of 47 residues and is the first example of a new subclass of plant defensins with five disulfide bonds whose structure has been determined. PhD1 has the fold of the cysteine-stabilized alphabeta motif, consisting of an alpha-helix and a triple-stranded antiparallel beta-sheet, except that it contains a fifth disulfide bond from the first loop to the alpha-helix. The additional disulfide bond is accommodated in PhD1 without any alteration of its tertiary structure with respect to other plant defensins. Comparison of its structure with those of classic, four-disulfide defensins has allowed us to identify a previously unrecognized hydrogen bond network that is integral to structure stabilization in the family.