Membrane-proximal Domain Research Articles

HLA-DM Stabilizes the Empty MHCII Binding Groove: A Model Using Customized Natural Move Monte Carlo.

MHC class II molecules bind peptides derived from extracellular proteins that have been ingested by antigen-presenting cells and display them to the immune system. Peptide loading occurs within the antigen-presenting cell and is facilitated by HLA-DM. HLA-DM stabilizes the open conformation of the MHCII binding groove when no peptide is bound. While a structure of the MHCII/HLA-DM complex exists, the mechanism of stabilization is still largely unknown. Here, we applied customized Natural Move Monte Carlo to investigate this interaction. We found a possible long-range mechanism that implicates the configuration of the membrane-proximal globular domains in stabilizing the open state of the empty MHCII binding groove.

ICA512 RESP18 homology domain is a protein-condensing factor and insulin fibrillation inhibitor

Type 1 diabetes islet cell autoantigen 512 (ICA512/IA-2) is a tyrosine phosphatase-like intrinsic membrane protein involved in the biogenesis and turnover of insulin secretory granules (SGs) in pancreatic islet β-cells. Whereas its membrane-proximal and cytoplasmic domains have been functionally and structurally characterized, the role of the ICA512 N-terminal segment named "regulated endocrine-specific protein 18 homology domain" (RESP18HD), which encompasses residues 35-131, remains largely unknown. Here, we show that ICA512 RESP18HD residues 91-131 encode for an intrinsically disordered region (IDR), which in vitro acts as a condensing factor for the reversible aggregation of insulin and other β-cell proteins in a pH and Zn2+-regulated fashion. At variance with what has been shown for other granule cargoes with aggregating properties, the condensing activity of ICA512 RESP18HD is displayed at a pH close to neutral, i.e. in the pH range found in the early secretory pathway, whereas it is resolved at acidic pH and Zn2+ concentrations resembling those present in mature SGs. Moreover, we show that ICA512 RESP18HD residues 35-90, preceding the IDR, inhibit insulin fibrillation in vitro Finally, we found that glucose-stimulated secretion of RESP18HD upon exocytosis of SGs from insulinoma INS-1 cells is associated with cleavage of its IDR, conceivably to prevent its aggregation upon exposure to neutral pH in the extracellular milieu. Taken together, these findings point to ICA512 RESP18HD being a condensing factor for protein sorting and granulogenesis early in the secretory pathway and for prevention of amyloidogenesis.

Role of KASH domain lengths in the regulation of LINC complexes.

The linker of the nucleoskeleton and cytoskeleton (LINC) complex is formed by the conserved interactions between Sad-1 and UNC-84 (SUN) and Klarsicht, ANC-1, SYNE homology (KASH) domain proteins, providing a physical coupling between the nucleoskeleton and cytoskeleton that mediates the transfer of physical forces across the nuclear envelope. The LINC complex can perform distinct cellular functions by pairing various KASH domain proteins with the same SUN domain protein. For example, in Caenorhabditis elegans, SUN protein UNC-84 binds to two KASH proteins UNC-83 and ANC-1 to mediate nuclear migration and anchorage, respectively. In addition to distinct cytoplasmic domains, the luminal KASH domain also varies among KASH domain proteins of distinct functions. In this study, we combined in vivo C. elegans genetics and in silico molecular dynamics simulations to understand the relation between the length and amino acid composition of the luminal KASH domain, and the function of the SUN–KASH complex. We show that longer KASH domains can withstand and transfer higher forces and interact with the membrane through a conserved membrane proximal EEDY domain that is unique to longer KASH domains. In agreement with our models, our in vivo results show that swapping the KASH domains of ANC-1 and UNC-83, or shortening the KASH domain of ANC-1, both result in a nuclear anchorage defect in C. elegans.

F-101 A hemagglutinin stalk-based universal influenza virus vaccine

Influenza virus infections are a significant cause of morbidity and mortality worldwide. Current vaccines show acceptable efficacy against antigenically matched viruses by inducing strain specific antibodies against the membrane-distal globular head domain of the viral hemagglutinin, but fail to protect against drifted and pandemic strains. The membrane-proximal stalk domain of the viral hemagglutinin exhibits a high degree of conservation across influenza virus subtypes, and monoclonal antibodies directed against this region typically show broad neutralizing activity. However, these antibodies are rare and usually not induced/boosted by regular seasonal vaccines. Here, we developed a universal influenza virus vaccination strategy based on the conserved stalk domain of group 1 and group 2 hemagglutinins. By sequential vaccination of mice and ferrets with chimeric hemagglutinin constructs that share the same stalk domain but have divergent head domains we were able to specifically boost broadly neutralizing antibody titers against conserved epitopes in the hemagglutinin stalk. Animals vaccinated with these constructs were protected from morbidity and mortality induced by infection with a panel of heterologous and heterosubtypic influenza A viruses, including avian influenza virus subtypes of concern like H5N1 and H7N9. The induced antibody response was long-lived and also interfered with virus transmission between animals. The present data suggest that this vaccine strategy has the potential to provide broad influenza virus protection in humans and clinical trials are currently ongoing. A universal influenza virus vaccine would represent a major advance towards the control of influenza worldwide and would significantly enhance our pandemic preparedness.

The Membrane Proximal Domain of TRPV1 and TRPV2 Channels Mediates Protein⁻Protein Interactions and Lipid Binding In Vitro.

Constitutive or regulated membrane protein trafficking is a key cell biology process. Transient receptor potential channels are somatosensory proteins in charge of detecting several physical and chemical stimuli, thus requiring fine vesicular trafficking. The membrane proximal or pre-S1 domain (MPD) is a highly conserved domain in transient receptor potential channels from the vanilloid (TRPV) subfamily. MPD shows traits corresponding to protein-protein and lipid-protein interactions, and protein regulatory regions. We have expressed MPD of TRPV1 and TRPV2 as green fluorescente protein (GFP)-fusion proteins to perform an in vitro biochemical and biophysical characterization. Pull-down experiments indicate that MPD recognizes and binds Soluble N-ethylmaleimide-sensitive factor Attachment Protein Receptors (SNARE). Synchrotron radiation scattering experiments show that this domain does not self-oligomerize. MPD interacts with phosphatidic acid (PA), a metabolite of the phospholipase D (PLD) pathway, in a specific manner as shown by lipid strips and Trp fluorescence quenching experiments. We show for the first time, to the best of our knowledge, the binding to PA of an N-terminus domain in TRPV channels. The presence of a PA binding domain in TRPV channels argues for putative PLD regulation. Findings in this study open new perspectives to understand the regulated and constitutive trafficking of TRPV channels exerted by protein-protein and lipid-protein interactions.

Length of KASH Domains Affect Linc Complex Functions

The linker of the nucleoskeleton and cytoskeleton (LINC) complexes are composed of inner nuclear membrane Sad1/UNC-84 (SUN, and outer nuclear membrane Klarsicht/ANC-1/SYNE homology (KASH) proteins. LINC complexes span the physical barrier of the nuclear envelope and regulate the physical connection between the interior of the nucleus and the cytoplasm during various cellular functions. The large cytoplasmic domains of KASH proteins bind to various elements of the cytoskeleton, whereas their 10-30 amino acid KASH domains reside in the perinuclear space (PNS) where they bind to SUN proteins. Interestingly, different KASH domain proteins can independently bind to the same SUN protein to mediate distinct cellular functions. For example, in mammals the SUN protein SUN1 can transiently associate with KASH5 in the PNS to mediate meiotic chromosome movement. Alternatively, SUN1 can bind to Nesprin1/2 to mediate nuclear movement. Similarly, in Caenorhabditis elegans (C. elegans) SUN protein UNC-84 transiently binds to KASH protein UNC-83 in embryonic hypodermal cells to mediate nuclear migration. Later, the same SUN protein UNC-84 independently binds to a different KASH protein ANC-1 to anchor of the nucleus in place for several days. In this work, we combined in vivo C. elegans genetics and in silico molecular dynamics simulations to explore the role of KASH domain length on the function of the SUN-KASH complex and its response to mechanical forces. Our results imply that longer KASH domains can withstand and transfer higher forces across the SUN-KASH complex. Additionally, our results suggest that longer KASH domains can interact with the membrane through a conserved membrane proximal EEDY domain that is unique to longer KASH domains. Finally, we show that swapping the KASH domains of ANC-1 and UNC-83, or shortening the KASH domain of ANC-1, both result in a nuclear anchorage defect in C. elegans.

Conformational Dynamics of T-Cell Receptor Chassis for Mechanosensing

AlphaBeta T-cell receptors (TCRαβ) are the major components of the adaptive immune system, sensing foreign antigens displayed on the surface of mammalian host cells altered by infection or other transformations with a remarkably high signal to noise ratio. Despite their high sensitivity, the affinity of each to the antigen peptide-bound major histocompatibility complex (pMHC) is low in thermal equilibrium, typically in the micromolar range. An emerging concept that reconciles the conundrum of high sensitivity versus low affinity is that the TCRαβ functions as a mechanosensor. Mechanical load on the TCRαβ-pMHC complex enhances the bond lifetime via activation of a catch bond pathway, a mechanism which is largely uncharacterized in structural terms. Past studies focused on behaviors of contacts directly involving TCRαβ and pMHC surfaces. However, such localized interactions alone cannot account for the catch bond since there is an allostery that modulates the catch bond behavior through TCR subdomains distal to the TCRαβ-pMHC interface. Thus, understanding of the conformational behavior of the entire TCR protein is required. Here we perform atomistic molecular dynamics simulations of TCRαβ as well as TCR gamma-delta (TCRγδ), receptors of cognate and innate T-cell immunity subsets, respectively. We also study a TCRγδ-αβ chimera that has the antigen-binding variable domains of TCRγδ with the membrane-proximal TCRαβ constant domains. By building ball-and-stick models of TCRs based on simulation trajectories, we examine relative motions of the four subdomains of each TCR type. We find that interdomain motion affects the organization of the pMHC-binding complementarity determining loops. These results suggest a TCR catch bond mechanism whereby an applied load alters the conformational dynamics of the subdomains and in turn, controls the TCR-pMHC interaction including resultant bond lifetime.

Anti-Human CD117 CAR T-Cells Efficiently Eliminate Hematopoietic Stem and CD117-Positive AML Cells

Introduction: Acute Myeloid Leukemia (AML) is a clonal disease of the hematopoietic system that originates from immature hematopoietic stem and progenitor cells (HSPC). Because some AML-initiating cells are comparatively resistant to conventional cytotoxic agents, disease relapses are common with current treatment approaches. As an alternative, immunological eradication of leukemic cells by adoptively transferred chimeric-antigen receptor T-cells (CAR T-cells) might be considerably more efficient. To date, however, the search for AML-specific surface antigens has remained largely elusive. To circumvent this problem, we propose to target the stem cell antigen c-Kit (CD117) that is expressed by physiological HSPC as wells as by leukemic blasts in >90% of AML patients. For translation into a clinical setting, CAR T cell treatment must then be followed by depletion of CAR T-cells as well subsequent healthy/allogeneic HSC transplantation.Methods: A lentiviral vector was generated which incorporates the CAR (scFv linked to intracellular CD3ζ and 4-1BB signaling domains via stalk and transmembrane regions derived from CD8), followed by a T2A ribosomal skip sequence and RQR8 as selection marker and depletion gene (surface expression of CD34 and CD20 epitopes). The scFv was extracted from a previously published bivalent anti-CD117 antibody (clone 79D) that was derived from an artificial human phage library (Reshetnyak et al., PNAS, 2013). 79D exhibits high binding affinity to an epitope in the membrane-proximal domain of human CD117. Human CD117 was cloned in human CD117 negative HL-60 AML cells and cell lines with stable expression of CD117 at various levels were derived from these.Results: T-cells were isolated from healthy donors or AML patients in complete remission and both healthy donor and AML pateint derived T-cells exhibited sustained growth after activation with recombinant human IL-2 and CD3/CD28 beads. Lentiviral transduction yielded consistently high transduction rates, ranging from 55 - 75% as determined by staining for RQR8 and the scFv.In co-culture assays, CAR T-cells eliminated more than 90% of CD117high leukemia cell lines within 24 hours at effector-to target ratios (E:T) of 4:1 and 1:1 and more than 50% at E:T of 1:4. CAR-mediated cytotoxicity correlated with levels of CD117 surface expression as the elimination of CD117low target cells was less efficient compared to CD117high and CD117intermediate cells. In long-term cytotoxicity assays (45d), only CD117low cells were able to escape CAR-mediated killing. In the setting of primary cells, anti-CD117 CAR T-cells effectively depleted >90% of lin-CD117+CD34+CD38+ and >70% of lin-CD117+CD34+CD38- cells from healthy bone marrow in vitro within 48 hours. Similarly, >70% of patient derived leukemic blasts were eliminated by autologous anti-CD117 CAR T-cells within 48 hours (1:1 ratio of CAR T cells:blasts). In a long-term assay, no outgrowth of leukemic blasts was observed in the presence of autologous CAR T-cells over 3 weeks.To determine effectivity of CAR T-cells in vivo, humanized mice (NSG & MTRG-SKI) were engrafted with umbilical cord blood derived CD34+ cells. A single injection of 2x106 anti-CD117 CAR T-cells resulted in >90% depletion of CD117+ cells in the bone marrow within 6 days. Finally, humanized mice transplanted with bone marrow from AML patients expressing CD117 were treated with patient-derived autologous CAR T-cells. At 6 weeks after injection of CAR T-cells, >98% of hu-CD45 CD117+ cells were depleted in the bone marrow while control human T-cell treated mice showed full-blown CD117 positive AML.Conclusions: We provide proof of concept for the generation of highly-potent CAR T-cells re-directed against CD117 from healthy human donors and AML patients. Anti-CD117 CAR T-cells exhibit high cytotoxic activity against CD117+ cell lines as well as primary healthy HSPC and patient AML cells in vitro and in vivo in murine xenograft models. Strategies for the complete elimination of CAR T-cells (immunologic or small molecule based) are required before translation of this approach to the clinical setting. DisclosuresNeri:Philochem AG: Equity Ownership.

Aggregatibacter actinomycetemcomitans leukotoxin causes activation of lymphocyte function-associated antigen 1.

Repeats-in-toxin leukotoxin (LtxA) produced by the oral bacterium Aggregatibacter actinomycetemcomitans kills human leukocytes in a lymphocyte function-associated antigen 1 (LFA-1, integrin αL /β2 )-dependent manner, although the mechanism for this interaction has not been identified. The LtxA internalisation by LFA-1-expressing cells was explored with florescence resonance energy transfer (FRET) microscopy using a cell line that expresses LFA-1 with a cyan fluorescent protein-tagged cytosolic αL domain and a yellow fluorescent protein-tagged β2 domain. Phorbol 12-myristate 13-acetate activation of LFA-1 caused transient cytosolic domain separation. However, addition of LtxA resulted in an increase in FRET, indicating that LtxA brings the cytosolic domains closer together, compared with the inactive state. Unlike activation, this effect was not transient, lasting more than 30min. Equilibrium constants of LtxA binding to the cytoplasmic domains of both αL and β2 were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the αL and β2 subunits (Kd =15 and 4.2nM, respectively) and a significantly lower affinity for the cytoplasmic domains of other integrin αM , αX , and β3 subunits (Kd =400, 180, and 230nM, respectively), used as controls. Peptide fragments of αL and β2 show that LtxA binds membrane-proximal domain of αL and intermediate domain of β2 .

The signalling conformation of the insulin receptor ectodomain

Understanding the structural biology of the insulin receptor and how it signals is of key importance in the development of insulin analogs to treat diabetes. We report here a cryo-electron microscopy structure of a single insulin bound to a physiologically relevant, high-affinity version of the receptor ectodomain, the latter generated through attachment of C-terminal leucine zipper elements to overcome the conformational flexibility associated with ectodomain truncation. The resolution of the cryo-electron microscopy maps is 3.2 Å in the insulin-binding region and 4.2 Å in the membrane-proximal region. The structure reveals how the membrane proximal domains of the receptor come together to effect signalling and how insulin’s negative cooperativity of binding likely arises. Our structure further provides insight into the high affinity of certain super-mitogenic insulins. Together, these findings provide a new platform for insulin analog investigation and design.

Structural insights into SorCS2\u2013Nerve Growth Factor complex formation

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.

A universal influenza virus vaccine strategy targeting the conserved stalk domain of the hemagglutinin

Influenza virus infections remain a significant cause of morbidity and mortality worldwide. Current vaccines show acceptable efficacy against antigenically matched viruses by inducing strain specific antibodies against the membrane-distal globular head domain of the viral hemagglutinin, but fail to protect against drifted and pandemic strains. The membrane-proximal stalk domain of the viral hemagglutinin exhibits a high degree of both sequence and structural conservation across influenza virus subtypes and monoclonal antibodies directed against this region typically show broad neutralizing activity. However, these antibodies are rare and usually not induced/boosted by regular seasonal vaccines. We hypothesize that a vaccine strategy that stimulates a robust immune response towards this region of the hemagglutinin could provide universal influenza virus protection. We developed a universal influenza virus vaccine based on the conserved stalk domain of group 1 and group 2 hemagglutinins. By sequential vaccination of mice and ferrets with chimeric hemagglutinin constructs that share the same stalk domain but have divergent head domains we were able to specifically boost broadly neutralizing antibody titers against conserved epitopes in the hemagglutinin stalk. Animals vaccinated with these constructs were protected from morbidity and mortality induced by infection with a panel of heterologous and heterosubtypic influenza A viruses. Additionally, chimeric hemagglutinin vaccination also impacted on virus transmission in the ferret model. In the light of emerging viruses in Asia it is of note that our vaccination regimen also protected animals from H5N1, H6N1 and H7N9 virus challenges and reduced lung titers upon H10 virus infection. Finally, we showed that stalk-reactive antibodies were boosted in individuals that received an H5N1 vaccine in clinical trials. This supports the hypothesis that exposure to hemagglutinins with divergent heads but conserved stalk induces such antibodies in humans. The present data suggest that this vaccine strategy has the potential to provide broad influenza virus protection in humans and clinical trials are currently ongoing. A universal influenza virus vaccine, which requires a single or only a few immunizations, would represent a major advance towards the control of influenza worldwide and would significantly enhance our pandemic preparedness.

Mechanotransduction by PCDH15 Relies on a Novel cis-Dimeric Architecture

The tip link, a filament formed by protocadherin 15 (PCDH15) and cadherin 23, conveys mechanical force from sound waves and head movement toopen hair-cell mechanotransduction channels. Tip-link cadherins are thought to have acquired structural features critical for their role in mechanotransduction. Here, we biophysically and structurally characterize the unusual cis-homodimeric architecture of PCDH15. We show that PCDH15 molecules form double-helical assemblies through cis-dimerization interfaces in the extracellular cadherin EC2-EC3 domain region and in a unique membrane-proximal domain. Electron microscopy studies visualize the cis-dimeric PCDH15 assembly and reveal the PCDH15 extracellular domain as a parallel double helix with cis cross-bridges at the two locations we defined. Thehelical configuration suggests the potential forelasticity through helix winding and unwinding. Functional studies in hair cells show that mutations that perturb PCDH15 dimerization contacts affect mechanotransduction. Together, these data reveal the cis-dimeric architecture of PCDH15 and show that dimerization is critical for sensing mechanical stimuli.

Abstract 2379: PTPRT pseudo-phosphatase domain is a denitrase that contributes to its tumor suppressor function

Abstract Department of Genetics & Genome Sciences and Case Comprehensive Cancer Center, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA. In addition to phosphorylation, tyrosine residues in proteins can have nitration at the 3-carbon position on the phenol ring (Y-NO2). Protein tyrosine nitration occurs under both physiological and pathologic conditions. However, enzymes that add or remove this protein modification remain to be identified. Protein tyrosine phosphatase receptor T (PTPRT) is one of the twelve receptor protein tyrosine phosphatases (RPTPs) that have two catalytic PTP domains in their intracellular parts. While the membrane proximal PTP domains (D1) are protein tyrosine phosphatases, it has been thought that the C-terminal PTP domains (D2) are pseudo-phosphatase that lack enzymatic activity. Here we report that the pseudo-phosphatase domain (D2) of PTPRT is a denitrase that removes nitro-groups from tyrosine residues in paxillin. PTPRT normally functions as a tumor suppressor and is frequently mutated in a variety of human cancers including colorectal cancer. Interestingly, a fifth of tumor-derived mutations of PTPRT are located in the D2 pseudo-PTP domain. We demonstrate that some of the tumor-derived mutations located in the pseudo-phosphatase domain impair the denitrase activity. Moreover, PTPRT mutant mice that inactivate the denitrase activity are susceptible to carcinogen-induced colon tumor formation. Our study uncovers a novel enzyme that functions as a tumor suppressor. Citation Format: Yiqing Zhao, Zhenghe Wang. PTPRT pseudo-phosphatase domain is a denitrase that contributes to its tumor suppressor function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2379.

CD6 monoclonal antibodies differ in epitope, kinetics and mechanism of action.

CD6 is a type I T-cell surface receptor that modulates antigen receptor signalling. Its activity is regulated by binding of its membrane proximal domain (domain 3) to a cell surface ligand, CD166. CD6 monoclonal antibodies (mAbs) specific for the membrane distal domain (domain 1) perturb CD6 function including itolizumab (Alzumab™), which has reached the clinic for treatment of autoimmune disease. We characterized molecular and functional properties of several CD6 mAbs including itolizumab to define potential mechanisms of action. Epitope mapping using the crystal structure of CD6 to design mutants identified two distinct binding sites on different faces of domain 1, one containing residue R77, crucial for MT605 and T12.1 binding and the other, E63, which is crucial for itolizumab and MEM98. Analysis of binding kinetics revealed that itolizumab has a lower affinity compared with other CD6 domain 1 mAbs. We compared potential agonistic (triggering) and antagonistic (blocking) properties of CD6 mAbs in assays where the mechanism of action was well defined. CD6 domain 1 and 3 mAbs were equally effective in triggering interleukin-2 production by a cell line expressing a chimeric antigen receptor containing the extracellular region of CD6. CD6 domain 1 mAbs hindered binding of multivalent immobilized CD166 but were inferior compared with blocking by soluble CD166 or a CD6 domain 3 mAb. Characterization of CD6 mAbs provides an insight into how their functional effects in vivo may be interpreted and their therapeutic use optimized.

NCAM2 Fibronectin type-III domains form a rigid structure that binds and activates the Fibroblast Growth Factor Receptor

NCAM1 and NCAM2 have ectodomains consisting of 5 Ig domains followed by 2 membrane-proximal FnIII domains. In this study we investigate and compare the structures and functions of these FnIII domains. The NCAM1 and -2 FnIII2 domains both contain a Walker A motif. In NCAM1 binding of ATP to this motif interferes with NCAM1 binding to FGFR. We obtained a structural model of the NCAM2 FnIII2 domain by NMR spectroscopy, and by titration with an ATP analogue we show that the NCAM2 Walker A motif does not bind ATP. Small angle X-ray scattering (SAXS) data revealed that the NCAM2 FnIII1-2 double domain exhibits a very low degree of flexibility. Moreover, recombinant NCAM2 FnIII domains bind FGFR in vitro, and the FnIII1-2 double domain induces neurite outgrowth in a concentration-dependent manner through activation of FGFR. Several synthetic NCAM1-derived peptides induce neurite outgrowth via FGFR. Only 2 of 5 peptides derived from similar regions in NCAM2 induce neurite outgrowth, but the most potent of these peptides stimulates neurite outgrowth through FGFR-dependent activation of the Ras-MAPK pathway. These results reveal that the NCAM2 FnIII domains form a rigid structure that binds and activates FGFR in a manner related to, but different from NCAM1.

Structural basis for membrane anchoring and fusion regulation of the herpes simplex virus fusogen gB.

Viral fusogens merge viral and cell membranes during cell penetration. Their ectodomains drive fusion by undergoing large-scale refolding, but little is known about the functionally important regions located within or near the membrane. Here, we report the crystal structure of the full-length glycoprotein B, the fusogen from Herpes Simplex Virus, complemented by electron spin resonance measurements. The membrane-proximal (MPR), transmembrane (TMD), and cytoplasmic (CTD) domains form a uniquely folded trimeric pedestal beneath the ectodomain, which balances dynamic flexibility with extensive, stabilizing membrane interactions. Hyperfusogenic mutations within the CTD destabilize it, targeting trimeric interfaces, structural motifs, and membrane-interacting elements. Thus, we propose that the CTD trimer observed in the structure stabilizes gB in its prefusion state despite being appended to the postfusion ectodomain. Our data suggest a model for how this dynamic, membrane-dependent “clamp” controls the fusogenic refolding of gB.

Characterization of a drug-targetable allosteric site regulating vascular endothelial growth factor signaling.

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases (VEGFRs). The extracellular domain of VEGFRs consists of seven Ig-homology domains, of which D2-3 form the ligand-binding site, while the membrane proximal domains D4-7 are involved in homotypic interactions in ligand-bound receptor dimers. Based on low-resolution structures, we identified allosteric sites in D4-5 and D7 of vascular endothelial growth factor receptor 2 (VEGFR-2) accomplishing regulatory functions. Allosteric inhibition of VEGFR-2 signaling represents an attractive option for the treatment of neovascular diseases. We showed earlier that DARPin® binders to domains D4 or D7 are potent VEGFR-2 inhibitors. Here we investigated in detail the allosteric inhibition mechanism of the domain D4 binding inhibitor D4b. The 2.38Å crystal structure of D4b in complex with VEGFR-2 D4-5, the first high-resolution structure of this VEGFR-2 segment, indicates steric hindrance by D4b as the mechanism of inhibition of receptor activation. At the cellular level, D4b triggered quantitative internalization of VEGFR-2 in the absence of ligand and thus clearance of VEGFR-2 from the surface of endothelial cells. The allosteric VEGFR-2 inhibition was sufficiently strong to efficiently inhibit the growth of human endothelial cells at suboptimal dose in a mouse xenograft model in vivo, underlining the therapeutic potential of the approach.

A Calmodulin C-Lobe Ca2+-Dependent Switch Governs Kv7 Channel Function

Kv7 (KCNQ) voltage-gated potassium channels control excitability in the brain, heart, and ear. Calmodulin (CaM) is crucial for Kv7 function, but how this calcium sensor affects activity has remained unclear. Here, we present X-ray crystallographic analysis of CaM:Kv7.4 and CaM:Kv7.5 AB domain complexes that reveal an Apo/CaM clamp conformation and calcium binding preferences. These structures, combined with small-angle X-ray scattering, biochemical, and functional studies, establish a regulatory mechanism for Kv7 CaM modulation based on a common architecture in which a CaM C-lobe calcium-dependent switch releases a shared Apo/CaM clamp conformation. This C-lobe switch inhibits voltage-dependent activation of Kv7.4 and Kv7.5 but facilitates Kv7.1, demonstrating that mechanism is shared by Kv7 isoforms despite the different directions of CaM modulation. Our findings provide a unified framework for understanding how CaM controls different Kv7 isoforms and highlight the role of membrane proximal domains for controlling voltage-gated channel function. VIDEO ABSTRACT.

Epsin and Sla2 form assemblies through phospholipid interfaces

In clathrin-mediated endocytosis, adapter proteins assemble together with clathrin through interactions with specific lipids on the plasma membrane. However, the precise mechanism of adapter protein assembly at the cell membrane is still unknown. Here, we show that the membrane–proximal domains ENTH of epsin and ANTH of Sla2 form complexes through phosphatidylinositol 4,5-bisphosphate (PIP2) lipid interfaces. Native mass spectrometry reveals how ENTH and ANTH domains form assemblies by sharing PIP2 molecules. Furthermore, crystal structures of epsin Ent2 ENTH domain from S. cerevisiae in complex with PIP2 and Sla2 ANTH domain from C. thermophilum illustrate how allosteric phospholipid binding occurs. A comparison with human ENTH and ANTH domains reveal only the human ENTH domain can form a stable hexameric core in presence of PIP2, which could explain functional differences between fungal and human epsins. We propose a general phospholipid-driven multifaceted assembly mechanism tolerating different adapter protein compositions to induce endocytosis.