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TREM2 is a signaling receptor expressed on microglia that has emerged as an important drug target for Alzheimer's disease and other neurodegenerative diseases. While a number of TREM2 ligands have been identified, little is known regarding the structural details of how they engage. To better understand this, we created a protein library of 28 different TREM2 variants that could be used to map interactions with various ligands using biolayer interferometry. The variants are located in previously identified putative binding surfaces on TREM2 called the hydrophobic site, basic site, and site 2. We found that mutations to the hydrophobic site ablated binding to apoE4 and TDP-43. Competition binding experiments indicated that apoE4 and oAβ42 share overlapping binding sites on TREM2. In contrast, binding to C1q was disrupted most strongly by mutations to the basic site, including R46, with some mutations to the hydrophobic site also attenuating binding, thus suggesting a broader mediation of binding across the two sites. Supporting this, competition experiments indicated that C1q binding could be blocked by both apoE and oAβ42. TREM2 binding to IL-34 was mediated by the basic site at a surface centering on R76. Competition binding experiments validated the unique site for IL-34, showing little to no competition with either oAβ42 or apoE4. However, competition experiments between C1q and IL34 suggest that the ligands compete for binding at the basic site. Altogether, our results suggest that TREM2 utilizes the hydrophobic site (consisting of CDR1, CDR2, and CDR3) as a common site to engage multiple ligands, and uses distinct basic sites to engage others. Our findings imply that pharmaceutical strategies targeting these surfaces might be effective to modulate TREM2 functions., Competing Interests: Declarations. Ethics approval and consent to participate: N/A. Competing interests: The authors report no competing interests., (© 2025. The Author(s).)

TREM2 is a signaling receptor expressed on microglia that has emerged as an important drug target for Alzheimer's disease and other neurodegenerative diseases. While a number of TREM2 ligands have been identified, little is known regarding the structural details of how they engage. To better understand this, we created a protein library of 28 different TREM2 variants that could be used to map interactions with various ligands using biolayer interferometry. The variants are located in previously identified putative binding surfaces on TREM2 called the hydrophobic site, basic site, and site 2. We found that mutations to the hydrophobic site ablated binding to apoE4 and TDP-43. Competition binding experiments indicated that apoE4 and oAβ42 share overlapping binding sites on TREM2. In contrast, binding to C1q was disrupted most strongly by mutations to the basic site, including R46, with some mutations to the hydrophobic site also attenuating binding, thus suggesting a broader mediation of binding across the two sites. Supporting this, competition experiments indicated that C1q binding could be blocked by both apoE and oAβ42. TREM2 binding to IL-34 was mediated by the basic site at a surface centering on R76. Competition binding experiments validated the unique site for IL-34, showing little to no competition with either oAβ42 or apoE4. However, competition experiments between C1q and IL34 suggest that the ligands compete for binding at the basic site. Altogether, our results suggest that TREM2 utilizes the hydrophobic site (consisting of CDR1, CDR2, and CDR3) as a common site to engage multiple ligands, and uses distinct basic sites to engage others. Our findings imply that pharmaceutical strategies targeting these surfaces might be effective to modulate TREM2 functions., Competing Interests: Competing Interests- the authors report no competing interests.

Introduction: The immune receptor triggering receptor expressed on myeloid cells 2 (TREM2) is among the strongest genetic risk factors for Alzheimer's disease (AD) and is a therapeutic target. TREM2 multimers have been identified in crystallography and implicated in the efficacy of antibody therapeutics; however, the molecular basis for TREM2 multimerization remains poorly understood., Methods: We used molecular dynamics simulations and binding energy analysis to determine the effects of AD-associated variants on TREM2 multimerization and validated with experimental results., Results: TREM2 trimers remained stably bound, driven primarily by salt bridge between residues D87 and R76 at the interface of TREM2 units. This salt bridge was disrupted by the AD-associated variants R47H and R98W and nearly ablated by the D87N variant. This decreased binding among TREM2 multimers was validated with co-immunoprecipitation assays., Discussion: This study uncovers a molecular basis for TREM2 forming stable trimers and unveils a novel mechanism by which TREM2 variants may increase AD risk by disrupting TREM2 oligomerization to impair TREM2 normal function., Highlights: Triggering receptor expressed on myeloid cells 2 (TREM2) multimerization could regulate TREM2 activation and function. D87-R76 salt bridges at the interface of TREM2 units drive the formation of stable TREM2 dimers and trimers. Alzheimer's disease (AD)-associated R47H and R98W variants disrupt the D87-R76 salt bridge. The AD-associated D87N variant leads to complete loss of the D87-R76 salt bridge., (© 2024 The Author(s). Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

The triggering receptor expressed on myeloid cells-2 (TREM2), a pivotal innate immune receptor, orchestrates functions such as inflammatory responses, phagocytosis, cell survival, and neuroprotection. TREM2 variants R47H and R62H have been associated with Alzheimer's disease, yet the underlying mechanisms remain elusive. Our previous research established that TREM2 binds to heparan sulfate (HS) and variants R47H and R62H exhibit reduced affinity for HS. Building upon this groundwork, our current study delves into the interplay between TREM2 and HS and its impact on microglial function. We confirm TREM2's binding to cell surface HS and demonstrate that TREM2 interacts with HS, forming HS-TREM2 binary complexes on microglia cell surfaces. Employing various biochemical techniques, including surface plasmon resonance, low molecular weight HS microarray screening, and serial HS mutant cell surface binding assays, we demonstrate TREM2's robust affinity for HS, and the effective binding requires a minimum HS size of approximately 10 saccharide units. Notably, TREM2 selectively binds specific HS structures, with 6-O-sulfation and, to a lesser extent, the iduronic acid residue playing crucial roles. N-sulfation and 2-O-sulfation are dispensable for this interaction. Furthermore, we reveal that 6-O-sulfation is essential for HS-TREM2 ternary complex formation on the microglial cell surface, and HS and its 6-O-sulfation are necessary for TREM2-mediated ApoE3 uptake in microglia. By delineating the interaction between HS and TREM2 on the microglial cell surface and demonstrating its role in facilitating TREM2-mediated ApoE uptake by microglia, our findings provide valuable insights that can inform targeted interventions for modulating microglial functions in Alzheimer's disease., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The Ca 2+ -activated Cl - channel regulator CLCA1 potentiates the activity of the Ca 2+ -activated Cl - channel (CaCC) TMEM16A by directly engaging the channel at the cell surface, inhibiting its reinternalization and increasing Ca 2+ -dependent Cl - current (I CaCC ) density. We now present evidence of functional pairing between two other CLCA and TMEM16 protein family members, namely CLCA4 and the CaCC TMEM16B. Similar to CLCA1, (i) CLCA4 is a self-cleaving metalloprotease, and the N-terminal portion (N-CLCA4) is secreted; (ii) the von Willebrand factor type A (VWA) domain in N-CLCA4 is sufficient to potentiate I CaCC in HEK293T cells; and (iii) this is mediated by the metal ion-dependent adhesion site motif within VWA. The results indicate that, despite the conserved regulatory mechanism and homology between CLCA1 and CLCA4, CLCA4-dependent I CaCC are carried by TMEM16B, rather than TMEM16A. Our findings show specificity in CLCA/TMEM16 interactions and suggest broad physiological and pathophysiological links between these two protein families., Competing Interests: Conflict of 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 Authors. Published by Elsevier Inc. All rights reserved.)

Milligram quantities of pure proteins are required for structural, functional, and pharmaceutical screening studies. These requirements can be challenging for a majority of important therapeutic targets that are secreted glycoproteins, receptors, membrane proteins, or large cytosolic complexes. Here, we present protocols for producing and purifying large amounts of secreted glycoproteins using the mammalian cell-based Expi293F system via large-scale transient transfection. This system can be easily adapted for the production of membrane proteins and large cytosolic complexes. The method can be utilized to quickly evaluate numerous expression constructs to identify optimal expressers. Use of mammalian cells ensures proper post-translational modifications, including disulfide bonds and glycosylation, that can be important for accurate functional studies. In addition, minor modifications can be introduced to produce labeled or deglycosylated proteins for structural studies by X-ray crystallography, nuclear magnetic resonance, or cryo-electron microscopy. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Production of milligram quantities of plasmid DNA for large-scale transient transfection Basic Protocol 2: Large-scale culture and transient transfection of Expi293F cells Basic Protocol 3: Purification of hexahistidine-tagged proteins from medium., (© 2022 Wiley Periodicals LLC.)

Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAP D3 ) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAP D3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections., Competing Interests: Declaration of interests H.W.V. is a founder of Casma Therapeutics and pierianDx and is employed by Vir Biotechnology. None of these companies funded the work reported here. Invention disclosures for method of use for Lrp1 interaction with RVFV Gn (G.K.A., A.L.H., D.W.L., H.W.V., and S.S.G.) and for the use of anti-Lrp1 antibodies by U Toronto (S.S.S., S.M., and G.K.A.) have been filed. Inclusion and diversity We worked to ensure sex balance in the selection of non-human subjects. We worked to ensure diversity in experimental samples through the selection of the cell lines. One or more of the authors of this paper self-identifies as an underrepresented ethnic minority in science. One or more of the authors of this paper self-identifies as a member of the LGBTQ+ community. While citing references scientifically relevant for this work, we also actively worked to promote gender balance in our reference list., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Introduction: Triggering receptor expressed on myeloid cells-2 (TREM2) is an immune receptor expressed on microglia that also can become soluble (sTREM2). How TREM2 engages different ligands remains poorly understood., Methods: We used comprehensive biolayer interferometry (BLI) analysis to investigate TREM2 and sTREM2 interactions with apolipoprotein E (apoE) and monomeric amyloid beta (Aβ) (mAβ42)., Results: TREM2 engagement of apoE was protein mediated with little effect of lipidation, showing slight affinity differences between isoforms (E4 > E3 > E2). Another family member, TREML2, did not bind apoE. Disease-linked TREM2 variants within a "basic patch" minimally impact apoE binding. Instead, TREM2 uses a unique hydrophobic surface to bind apoE, which requires the apoE hinge region. TREM2 and sTREM2 directly bind mAβ42 and potently inhibit Aβ42 polymerization, suggesting a potential role for soluble sTREM2 in preventing AD pathogenesis., Discussion: These findings demonstrate that TREM2 has at least two ligand-binding surfaces that might be therapeutic targets and uncovers a potential function for sTREM2 in directly inhibiting Aβ polymerization., (© 2020 the Alzheimer's Association.)

The secreted protein calcium-activated chloride channel regulator 1 (CLCA1) utilizes a von Willebrand factor type A (VWA) domain to bind to and potentiate the calcium-activated chloride channel TMEM16A. To gain insight into this unique potentiation mechanism, we determined the 2.0-Å crystal structure of human CLCA1 VWA bound to Ca 2+ . The structure reveals the metal-ion-dependent adhesion site (MIDAS) in a high-affinity "open" conformation, engaging in crystal contacts that likely mimic how CLCA1 engages TMEM16A. The CLCA1 VWA contains a disulfide bond between α3 and α4 in close proximity to the MIDAS that is invariant in the CLCA family and unique in VWA structures. Further biophysical studies indicate that CLCA1 VWA is preferably stabilized by Mg 2+ over Ca 2+ and that α6 atypically extends from the VWA core. Finally, an analysis of TMEM16A structures suggests residues likely to mediate interaction with CLCA1 VWA., Competing Interests: Declaration of Interests The authors declare no conflicts of interest., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Soluble triggering receptor expressed on myeloid cells 2 (sTREM2) in cerebrospinal fluid (CSF) has been associated with Alzheimer's disease (AD). TREM2 plays a critical role in microglial activation, survival, and phagocytosis; however, the pathophysiological role of sTREM2 in AD is not well understood. Understanding the role of sTREM2 in AD may reveal new pathological mechanisms and lead to the identification of therapeutic targets. We performed a genome-wide association study (GWAS) to identify genetic modifiers of CSF sTREM2 obtained from the Alzheimer's Disease Neuroimaging Initiative. Common variants in the membrane-spanning 4-domains subfamily A ( MS4A ) gene region were associated with CSF sTREM2 concentrations (rs1582763; P = 1.15 × 10 -15 ); this was replicated in independent datasets. The variants associated with increased CSF sTREM2 concentrations were associated with reduced AD risk and delayed age at onset of disease. The single-nucleotide polymorphism rs1582763 modified expression of the MS4A4A and MS4A6A genes in multiple tissues, suggesting that one or both of these genes are important for modulating sTREM2 production. Using human macrophages as a proxy for microglia, we found that MS4A4A and TREM2 colocalized on lipid rafts at the plasma membrane, that sTREM2 increased with MS4A4A overexpression, and that silencing of MS4A4A reduced sTREM2 production. These genetic, molecular, and cellular findings suggest that MS4A4A modulates sTREM2. These findings also provide a mechanistic explanation for the original GWAS signal in the MS4A locus for AD risk and indicate that TREM2 may be involved in AD pathogenesis not only in TREM2 risk-variant carriers but also in those with sporadic disease., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Members of Mononegavirales, the order that includes nonsegmented negative sense RNA viruses (NNSVs), encode a small number of multifunctional proteins. In members of the Filoviridae family, virus protein 35 (VP35) facilitates immune evasion and functions as an obligatory cofactor for viral RNA synthesis. VP35 functions in a manner orthologous to that of phosphoproteins from other NNSVs. Although the critical roles of Ebola viral VP35 (eVP35) in immune evasion and RNA synthesis are well-appreciated, a complete understanding of its organization and its role in carrying out its many functions has yet to be fully realized. In particular, we currently lack information about the role of the oligomerization domain within eVP35. To address this limitation, we report here an investigation of the oligomer structure of eVP35 using hybrid methods that include multiangle light scattering, small-angle X-ray scattering, and cross-linking coupled with mass spectrometry to determine the shape and orientation of the eVP35 oligomer. Our integrative results are consistent with a parallel tetramer in which the N-terminal regions that are required for RNA synthesis are all oriented in the same direction. Furthermore, these results define a framework for targeting the symmetric tetramer for structure-based antiviral discovery.

Clinical isolates of Yersinia , Klebsiella , and Escherichia coli frequently secrete the small molecule metallophore yersiniabactin (Ybt), which passivates and scavenges transition metals during human infections. YbtT is encoded within the Ybt biosynthetic operon and is critical for full Ybt production in bacteria. However, its biosynthetic function has been unclear because it is not essential for Ybt production by the in vitro reconstituted nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway. Here, we report the structural and biochemical characterization of YbtT. YbtT structures at 1.4-1.9 Å resolution possess a serine hydrolase catalytic triad and an associated substrate chamber with features similar to those previously reported for low-specificity type II thioesterases (TEIIs). We found that YbtT interacts with the two major Ybt biosynthetic proteins, HMWP1 (high-molecular-weight protein 1) and HMWP2 (high-molecular-weight protein 2), and hydrolyzes a variety of aromatic and acyl groups from their phosphopantetheinylated carrier protein domains. In vivo YbtT titration in uropathogenic E. coli revealed a distinct optimum for Ybt production consistent with a tradeoff between clearing both stalled inhibitory intermediates and productive Ybt precursors from HMWP1 and HMWP2. These results are consistent with a model in which YbtT maintains cellular Ybt biosynthesis by removing nonproductive, inhibitory thioesters that form aberrantly at multiple sites on HMWP1 and HMWP2., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Recent findings regarding the cellular biology and immunology of BST-2 (also known as tetherin) indicate that its function could be exploited as a universal replication inhibitor of enveloped respiratory viruses (e.g., influenza, respiratory syncytial virus, etc.). BST-2 inhibits viral replication by preventing virus budding from the plasma membrane and by inducing an antiviral state in cells adjacent to infection via unique inflammatory signaling mechanisms. This review presents the first comprehensive summary of what is currently known about BST-2 anti-viral function against respiratory viruses, how these viruses construct countermeasures to antagonize BST-2, and how BST-2 function might be targeted to develop therapies to treat respiratory virus infections. The authors address the current gaps in knowledge, including the need for mechanistic understanding of BST-2 antagonism by respiratory viruses, that should be bridged to achieve that goal., (© 2018 WILEY Periodicals, Inc.)

The protein triggering receptor expressed on myeloid cells-2 (TREM2) is an immunomodulatory receptor with a central role in myeloid cell activation and survival. In recent years, the importance of TREM2 has been highlighted by the identification of coding variants that increase risk for Alzheimer's disease and other neurodegenerative diseases. Animal studies have further shown the importance of TREM2 in neurodegenerative and other inflammatory disease models including chronic obstructive pulmonary disease, multiple sclerosis, and stroke. A mechanistic understanding of TREM2 function remains elusive, however, due in part to the absence of conclusive information regarding the identity of endogenous TREM2 ligands. While many TREM2 ligands have been proposed, their physiological role and mechanism of engagement remain to be determined. In this review, we highlight the suggested roles of TREM2 in these diseases and the recent advances in our understanding of TREM2 and discuss putative TREM2-ligand interactions and their potential roles in signaling during health and disease. We develop a model based on the TREM2 structure to explain how different TREM2 ligands might interact with the receptor and how disease risk variants may alter ligand interactions. Finally, we propose future experimental directions to establish the role and importance of these different interactions on TREM2 function., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Calcium-activated chloride channels (CaCCs) are key players in transepithelial ion transport and fluid secretion, smooth muscle constriction, neuronal excitability, and cell proliferation. The CaCC regulator 1 (CLCA1) modulates the activity of the CaCC TMEM16A/Anoctamin 1 (ANO1) by directly engaging the channel at the cell surface, but the exact mechanism is unknown. Here we demonstrate that the von Willebrand factor type A (VWA) domain within the cleaved CLCA1 N-terminal fragment is necessary and sufficient for this interaction. TMEM16A protein levels on the cell surface were increased in HEK293T cells transfected with CLCA1 constructs containing the VWA domain, and TMEM16A-like currents were activated. Similar currents were evoked in cells exposed to secreted VWA domain alone, and these currents were significantly knocked down by TMEM16A siRNA. VWA-dependent TMEM16A modulation was not modified by the S357N mutation, a VWA domain polymorphism associated with more severe meconium ileus in cystic fibrosis patients. VWA-activated currents were significantly reduced in the absence of extracellular Mg 2+ , and mutation of residues within the conserved metal ion-dependent adhesion site motif impaired the ability of VWA to potentiate TMEM16A activity, suggesting that CLCA1-TMEM16A interactions are Mg 2+ - and metal ion-dependent adhesion site-dependent. Increase in TMEM16A activity occurred within minutes of exposure to CLCA1 or after a short treatment with nocodazole, consistent with the hypothesis that CLCA1 stabilizes TMEM16A at the cell surface by preventing its internalization. Our study hints at the therapeutic potential of the selective activation of TMEM16A by the CLCA1 VWA domain in loss-of-function chloride channelopathies such as cystic fibrosis., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Genetic variations in the myeloid immune receptor TREM2 are linked to several neurodegenerative diseases. To determine how TREM2 variants contribute to these diseases, we performed structural and functional studies of wild-type and variant proteins. Our 3.1 Å TREM2 crystal structure revealed that mutations found in Nasu-Hakola disease are buried whereas Alzheimer's disease risk variants are found on the surface, suggesting that these mutations have distinct effects on TREM2 function. Biophysical and cellular methods indicate that Nasu-Hakola mutations impact protein stability and decrease folded TREM2 surface expression, whereas Alzheimer's risk variants impact binding to a TREM2 ligand. Additionally, the Alzheimer's risk variants appear to epitope map a functional surface on TREM2 that is unique within the larger TREM family. These findings provide a guide to structural and functional differences among genetic variants of TREM2, indicating that therapies targeting the TREM2 pathway should be tailored to these genetic and functional differences with patient-specific medicine approaches for neurodegenerative disorders., Competing Interests: The authors declare that no competing interests exist.

Background: P38 MAP kinases are centrally involved in mediating extracellular signaling in various diseases. While much attention has previously been focused on the ubiquitously expressed family member MAPK14 (p38α), recent studies indicate that family members such as MAPK13 (p38δ) display a more selective cellular and tissue expression and might therefore represent a specific kinase to target in certain diseases., Methods: To facilitate the design of potent and specific inhibitors, we present here the structural, biophysical, and functional characterization of two new MAPK13-inhibitor complexes, as well as the first comprehensive structural, biophysical, and functional analysis of MAPK13 complexes with four different inhibitor compounds of greatly varying potency., Results: These inhibitors display IC50 values either in the nanomolar range or micromolar range (>800-fold range). The nanomolar inhibitors exhibit much longer ligand-enzyme complex half-lives compared to the micromolar inhibitors as measured by biolayer interferometry. Crystal structures of the MAPK13 inhibitor complexes reveal that the nanomolar inhibitors engage MAPK13 in the DFG-out binding mode, while the micromolar inhibitors are in the DFG-in mode. Detailed structural and computational docking analyses suggest that this difference in binding mode engagement is driven by conformational restraints imposed by the chemical structure of the inhibitors, and may be fortified by an additional hydrogen bond to MAPK13 in the nanomolar inhibitors., Conclusions: These studies provide a structural basis for understanding the differences in potency exhibited by these inhibitors., General Significance: They also provide the groundwork for future studies to improve specificity, potency, pharmacodynamics, and pharmacokinetic properties., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background and Purpose: TMEM16A, also known as anoctamin 1 channel, is a member of the Ca(2+)-activated chloride channels family and serves as a heat sensor in the primary nociceptors. Eact is a recently discovered small molecule activator of the TMEM16A channel. Here, we asked if Eact produces pain- and itch-related responses in vivo and investigated the cellular and molecular basis of Eact-elicited responses in dorsal root ganglia (DRG) neurons., Experimental Approach: We employed behavioural testing combined with pharmacological inhibition and genetic ablation approaches to identify transient receptor potential vanilloid 1 (TRPV1) as the prominent mediator for Eact-evoked itch- or pain-related responses. We investigated the effects of Eact on TRPV1 and TMEM16A channels expressed in HEK293T cells and in DRG neurons isolated from wild type and Trpv1(-/-) mice using Ca(2+) imaging and patch-clamp recordings. We also used site-directed mutagenesis to determine the molecular basis of Eact activation of TRPV1., Key Results: Administration of Eact elicited both itch- and pain-related behaviours. Unexpectedly, the Eact-elicited behavioural responses were dependent on the function of TRPV1, as shown by pharmacological inhibition and genetic ablation studies. Eact activated membrane currents and increased intracellular free Ca(2+) in both TRPV1-expressing HEK293T cells and isolated DRG neurons in a TRPV1-dependent manner. Eact activation of the TRPV1 channel was severely attenuated by mutations disrupting the capsaicin-binding sites., Conclusions and Implications: Our results suggest that Eact activates primary sensory nociceptors and produces both pain and itch responses mainly through direct activation of TRPV1 channels., (© 2016 The British Pharmacological Society.)

Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis.

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve hyper-responsiveness to interferon without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depended on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acted on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to degrade these proteases via the immunoproteasome. Thus, PARP9-DTX3L acted on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway.

Viral infections and type 2 immune responses are thought to be critical for the development of chronic respiratory disease, but the link between these events needs to be better defined. Here, we study a mouse model in which infection with a mouse parainfluenza virus known as Sendai virus (SeV) leads to long-term activation of innate immune cells that drive IL-13-dependent lung disease. We find that chronic postviral disease (signified by formation of excess airway mucus and accumulation of M2-differentiating lung macrophages) requires macrophage expression of triggering receptor expressed on myeloid cells-2 (TREM-2). Analysis of mechanism shows that viral replication increases lung macrophage levels of intracellular and cell surface TREM-2, and this action prevents macrophage apoptosis that would otherwise occur during the acute illness (5-12 d after inoculation). However, the largest increases in TREM-2 levels are found as the soluble form (sTREM-2) long after clearance of infection (49 d after inoculation). At this time, IL-13 and the adapter protein DAP12 promote TREM-2 cleavage to sTREM-2 that is unexpectedly active in preventing macrophage apoptosis. The results thereby define an unprecedented mechanism for a feed-forward expansion of lung macrophages (with IL-13 production and consequent M2 differentiation) that further explains how acute infection leads to chronic inflammatory disease., (© 2015 Wu et al.)

The p38 MAP kinases (p38 MAPKs) represent an important family centrally involved in mediating extracellular signaling. Recent studies indicate that family members such as MAPK13 (p38δ) display a selective cellular and tissue expression and are therefore involved in specific diseases. Detailed structural studies of all p38 MAPK family members are crucial for the design of specific inhibitors. In order to facilitate such ventures, the structure of MAPK13 was determined in both the inactive (unphosphorylated; MAPK13) and active (dual phosphorylated; MAPK13/pTpY) forms. Here, the first preparation, crystallization and structure determination of MAPK13/pTpY are presented and the structure is compared with the previously reported structure of MAPK13 in order to facilitate studies for structure-based drug design. A comprehensive analysis of inactive versus active structures for the p38 MAPK family is also presented. It is found that MAPK13 undergoes a larger interlobe configurational rearrangement upon activation compared with MAPK14. Surprisingly, the analysis of activated p38 MAPK structures (MAP12/pTpY, MAPK13/pTpY and MAPK14/pTpY) reveals that, despite a high degree of sequence similarity, different side chains are used to coordinate the phosphorylated residues. There are also differences in the rearrangement of the hinge region that occur in MAPK14 compared with MAPK13 which would affect inhibitor binding. A thorough examination of all of the active (phosphorylated) and inactive (unphosphorylated) p38 MAPK family member structures was performed to reveal a common structural basis of activation for the p38 MAP kinase family and to identify structural differences that may be exploited for developing family member-specific inhibitors.

Calcium-activated chloride channel regulator 1 (CLCA1) activates calcium-dependent chloride currents; neither the target, nor mechanism, is known. We demonstrate that secreted CLCA1 activates calcium-dependent chloride currents in HEK293T cells in a paracrine fashion, and endogenous TMEM16A/Anoctamin1 conducts the currents. Exposure to exogenous CLCA1 increases cell surface levels of TMEM16A and cellular binding experiments indicate CLCA1 engages TMEM16A on the surface of these cells. Altogether, our data suggest that CLCA1 stabilizes TMEM16A on the cell surface, thus increasing surface expression, which results in increased calcium-dependent chloride currents. Our results identify the first Cl(-) channel target of the CLCA family of proteins and establish CLCA1 as the first secreted direct modifier of TMEM16A activity, delineating a unique mechanism to increase currents. These results suggest cooperative roles for CLCA and TMEM16 proteins in influencing the physiology of multiple tissues, and the pathology of multiple diseases, including asthma, COPD, cystic fibrosis, and certain cancers.

Chloride transport proteins play critical roles in inflammatory airway diseases, contributing to the detrimental aspects of mucus overproduction, mucus secretion, and airway constriction. However, they also play crucial roles in contributing to the innate immune properties of mucus and mucociliary clearance. In this review, we focus on the emerging novel roles for a chloride channel regulator (CLCA1), a calcium-activated chloride channel (TMEM16A), and two chloride exchangers (SLC26A4/pendrin and SLC26A9) in chronic inflammatory airway diseases.

During antiviral defense, interferon (IFN) signaling triggers nuclear transport of tyrosine-phosphorylated STAT1 (PY-STAT1), which occurs via a subset of karyopherin alpha (KPNA) nuclear transporters. Many viruses, including Ebola virus, actively antagonize STAT1 signaling to counteract the antiviral effects of IFN. Ebola virus VP24 protein (eVP24) binds KPNA to inhibit PY-STAT1 nuclear transport and render cells refractory to IFNs. We describe the structure of human KPNA5 C terminus in complex with eVP24. In the complex, eVP24 recognizes a unique nonclassical nuclear localization signal (NLS) binding site on KPNA5 that is necessary for efficient PY-STAT1 nuclear transport. eVP24 binds KPNA5 with very high affinity to effectively compete with and inhibit PY-STAT1 nuclear transport. In contrast, eVP24 binding does not affect the transport of classical NLS cargo. Thus, eVP24 counters cell-intrinsic innate immunity by selectively targeting PY-STAT1 nuclear import while leaving the transport of other cargo that may be required for viral replication unaffected., (Copyright © 2014 Elsevier Inc. All rights reserved.)

TREM-2 (triggering receptor expressed on myeloid cells-2) is an innate immune receptor expressed on dendritic cells, macrophages, osteoclasts, and microglia. Recent genetic studies have reported the occurrence of point mutations in TREM-2 that correlate with a dramatically increased risk for the development of neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia, and Parkinson's disease. Structural and biophysical studies of wild-type and mutant TREM-2 ectodomains are required to understand the functional consequences of these mutations. In order to facilitate these studies, we undertook the production and crystallization of these proteins. Here we demonstrate that, unlike many single Ig domain proteins, TREM-2 could not be readily refolded from bacterially-expressed inclusion bodies. Instead, we developed a mammalian-cell based expression system for the successful production of wild-type and mutant TREM-2 proteins in milligram quantities and a single-chromatography-step purification scheme that produced diffraction-quality crystals. These crystals diffract to a resolution of 3.3 Å and produce data sufficient for structure determination. We describe herein the procedures to produce wild-type and mutant human TREM-2 Ig domains in sufficient quantities for structural and biophysical studies. Such studies are crucial to understand the functional consequences of TREM-2 point mutations linked to the development of neurodegenerative diseases and, ultimately, to develop patient-specific molecular therapies to treat them., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Microfibril-associated glycoprotein (MAGP) 1 and 2 are evolutionarily related but structurally divergent proteins that are components of microfibrils of the extracellular matrix. Using mice with a targeted inactivation of Mfap5, the gene for MAGP2 protein, we demonstrate that MAGPs have shared as well as unique functions in vivo. Mfap5(-/-) mice appear grossly normal, are fertile, and have no reduction in life span. Cardiopulmonary development is typical. The animals are normotensive and have vascular compliance comparable with age-matched wild-type mice, which is indicative of normal, functional elastic fibers. Loss of MAGP2 alone does not significantly alter bone mass or architecture, and loss of MAGP2 in tandem with loss of MAGP1 does not exacerbate MAGP1-dependent osteopenia. MAGP2-deficient mice are neutropenic, which contrasts with monocytopenia described in MAGP1-deficient animals. This suggests that MAGP1 and MAGP2 have discrete functions in hematopoiesis. In the cardiovascular system, MAGP1;MAGP2 double knockout mice (Mfap2(-/-);Mfap5(-/-)) show age-dependent aortic dilation. These findings indicate that MAGPs have shared primary functions in maintaining large vessel integrity. In solid phase binding assays, MAGP2 binds active TGFβ1, TGFβ2, and BMP2. Together, these data demonstrate that loss of MAGP2 expression in vivo has pleiotropic effects potentially related to the ability of MAGP2 to regulate growth factors or participate in cell signaling.

BST-2 (also known as tetherin, CD317, or HM1.24) was first described as a potent interferon-inducible host antiviral factor nearly five years ago. Since that time, numerous reports have been published regarding the antiviral activity and immunological properties of this protein. BST-2 blocks viral replication by inhibiting enveloped virus budding from the surface of infected cells. To counteract this, most viruses have developed strategies to antagonize BST-2, each employing a unique mechanism. In this review, we summarize the antiviral function, structural biology and immunobiology of BST-2. Taken together, our current understanding of BST-2 suggests potential avenues as well as challenges to exploiting its action in the development of broad spectrum antiviral treatments., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The chloride channel calcium-activated (CLCA) family are secreted proteins that regulate both chloride transport and mucin expression, thus controlling the production of mucus in respiratory and other systems. Accordingly, human CLCA1 is a critical mediator of hypersecretory lung diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, that manifest mucus obstruction. Despite relevance to homeostasis and disease, the mechanism of CLCA1 function remains largely undefined. We address this void by showing that CLCA proteins contain a consensus proteolytic cleavage site recognized by a novel zincin metalloprotease domain located within the N terminus of CLCA itself. CLCA1 mutations that inhibit self-cleavage prevent activation of calcium-activated chloride channel (CaCC)-mediated chloride transport. CaCC activation requires cleavage to unmask the N-terminal fragment of CLCA1, which can independently gate CaCCs. Gating of CaCCs mediated by CLCA1 does not appear to involve proteolytic cleavage of the channel because a mutant N-terminal fragment deficient in proteolytic activity is able to induce currents comparable with that of the native fragment. These data provide both a mechanistic basis for CLCA1 self-cleavage and a novel mechanism for regulation of chloride channel activity specific to the mucosal interface.

Increased mucus production is a common cause of morbidity and mortality in inflammatory airway diseases, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. However, the precise molecular mechanisms for pathogenic mucus production are largely undetermined. Accordingly, there are no specific and effective anti-mucus therapeutics. Here, we define a signaling pathway from chloride channel calcium-activated 1 (CLCA1) to MAPK13 that is responsible for IL-13-driven mucus production in human airway epithelial cells. The same pathway was also highly activated in the lungs of humans with excess mucus production due to COPD. We further validated the pathway by using structure-based drug design to develop a series of novel MAPK13 inhibitors with nanomolar potency that effectively reduced mucus production in human airway epithelial cells. These results uncover and validate a new pathway for regulating mucus production as well as a corresponding therapeutic approach to mucus overproduction in inflammatory airway diseases.

The GGA family of clathrin adaptor proteins mediates the intracellular trafficking of transmembrane proteins by interacting with DXXLL-type sorting signals on the latter. These signals were originally identified at the carboxy-termini of the transmembrane cargo proteins. Subsequent studies, however, showed that internal DXXLL sorting motifs occur within the N- or C-terminal cytoplasmic domains of cargo molecules. The GGAs themselves also contain internal DXXLL motifs that serve to auto-regulate GGA function. A recent study challenged the notion that internal DXXLL signals are competent for binding to GGAs. Since the question of whether GGA adaptors interact with internal DXXLL motifs is fundamental to the identification of bona fide GGA cargo, and to an accurate understanding of GGA regulation within cells, we have extended our previous findings. We now present additional evidence confirming that GGAs do interact with internal DXXLL motifs. We also summarize the recent reports from other laboratories documenting internal GGA binding motifs., (© 2012 John Wiley & Sons A/S.)

Tropoelastin (TE), the soluble monomer of elastin, is synthesized by elastogenic cells, such as chondrocytes, fibroblasts, and smooth muscle cells (SMCs). The C-terminal domain of TE interacts with cell receptors, and these interactions play critical roles in elastic fiber assembly. We recently found that oxidation of TE prevents elastic fiber assembly. Here, we examined the effects of oxidation of TE on cell interactions. We found that SMCs bind to TE through heparan sulfate (HS), whereas fetal lung fibroblasts (WI-38 cells) bind through integrin α(v)β(3) and HS. In addition, we found that oxidation of TE by peroxynitrite (ONOO(-)) prevented binding of SMCs and WI-38 cells and other elastogenic cells, human dermal fibroblasts and fetal bovine chondrocytes. Because the C-terminal domain of TE has binding sites for both HS and integrin, we examined the effects of oxidation of a synthetic peptide derived from the C-terminal 25 amino acids of TE (CT-25) on cell binding. The CT-25 peptide contains the only two Cys residues in TE juxtaposed to a cluster of positively charged residues (RKRK) that are important for cell binding. ONOO(-) treatment of the CT-25 peptide prevented cell binding, whereas reduction of the CT-25 peptide had no effect. Mass spectrometric and circular dichroism spectroscopic analyses showed that ONOO(-) treatment modified both Cys residues in the CT-25 peptide to sulfonic acid derivatives, without altering the secondary structure. These data suggest that the mechanism by which ONOO(-) prevents cell binding to TE is by introducing negatively charged sulfonic acid residues near the positively charged cluster.

BST-2/tetherin is a host antiviral molecule that functions to potently inhibit the release of enveloped viruses from infected cells. In return, viruses have evolved antagonists to this activity. BST-2 traps budding virions by using two separate membrane-anchoring regions that simultaneously incorporate into the host and viral membranes. Here, we detailed the structural and biophysical properties of the full-length BST-2 ectodomain, which spans the two membrane anchors. The 1.6-Å crystal structure of the complete mouse BST-2 ectodomain reveals an ∼145-Å parallel dimer in an extended α-helix conformation that predominantly forms a coiled coil bridged by three intermolecular disulfides that are required for stability. Sequence analysis in the context of the structure revealed an evolutionarily conserved design that destabilizes the coiled coil, resulting in a labile superstructure, as evidenced by solution x-ray scattering displaying bent conformations spanning 150 and 180 Å for the mouse and human BST-2 ectodomains, respectively. Additionally, crystal packing analysis revealed possible curvature-sensing tetrameric structures that may aid in proper placement of BST-2 during the genesis of viral progeny. Overall, this extended coiled-coil structure with inherent plasticity is undoubtedly necessary to accommodate the dynamics of viral budding while ensuring separation of the anchors.

Inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) exhibit stereotyped traits that are variably expressed in each person. In experimental mouse models of chronic lung disease, these individual disease traits can be genetically segregated and thereby linked to distinct determinants. Functional genomic analysis indicates that at least one of these traits, mucous cell metaplasia, depends on members of the calcium-activated chloride channel (CLCA) gene family. Here we review advances in the biochemistry of the CLCA family and the evidence of a role for CLCA family members in the development of mucous cell metaplasia and possibly airway hyperreactivity in experimental models and in humans. On the basis of this information, we develop the model that CLCA proteins are not integral membrane proteins with ion channel function but instead are secreted signaling molecules that specifically regulate airway target cells in healthy and disease conditions.

Endocytic clathrin-coated vesicles arise through the deformation of a small region of plasma membrane encapsulated by a cytosol-oriented clathrin lattice. The coat assembles from soluble protomers in a rapid and highly cooperative process, and invagination is tightly linked to the selective enrichment of cargo molecules within the nascent bud. Recent structural and functional studies demonstrate that coat assembly, membrane deformation, local actin dynamics and the final scission event are intricately coupled, and begin to reveal how key multifunctional, modular proteins are responsible for this linkage. An emerging mechanistic theme is how sequential engagement of common interaction surfaces or network hubs can evict prior binding partners from the assembly zone to ensure vectorial progression of the coat assembly process.

Huntingtin-interacting protein-1 related (HIP1R) has a crucial protein-trafficking role, mediating associations between actin and clathrin-coated structures at the plasma membrane and trans-Golgi network. Here, we characterize the F-actin-binding region of HIP1R, termed the talin-HIP1/R/Sla2p actin-tethering C-terminal homology (THATCH) domain. The 1.9-A crystal structure of the human HIP1R THATCH core reveals a large sequence-conserved surface patch created primarily by residues from the third and fourth helices of a unique five-helix bundle. Point mutations of seven contiguous patch residues produced significant decreases in F-actin binding. We also show that THATCH domains have a conserved C-terminal latch capable of oligomerizing the core, thereby modulating F-actin engagement. Collectively, these results establish a framework for investigating the links between endocytosis and actin dynamics mediated by THATCH domain-containing proteins.

Clathrin-mediated endocytosis depends upon the coordinated assembly of a large number of discrete clathrin coat components to couple cargo selection with rapid internalization from the cell surface. Accordingly, the heterotetrameric AP-2 adaptor complex binds not only to clathrin and select cargo molecules, but also to an extensive family of endocytic accessory factors and alternate sorting adaptors. Physical associations between accessory proteins and AP-2 occur primarily through DP(F/W) or FXDXF motifs, which engage an interaction surface positioned on the C-terminal platform subdomain of the independently folded alpha subunit appendage. Here, we find that the WXX(F/W)X(D/E) interaction motif found in several endocytic proteins, including synaptojanin 1, stonin 2, AAK1, GAK, and NECAP1, binds a second interaction site on the bilobal alpha appendage, located on the N-terminal beta sandwich subdomain. Both alpha appendage binding sites can be engaged synchronously, and our data reveal that varied assemblies of interaction motifs with different affinities for two sites upon the alpha appendage can provide a mechanism for temporal ordering of endocytic accessory proteins during clathrin-mediated endocytosis.

The N-terminal protein interaction domain (N-domain) of the signal transducer and activator of transcription-4 (STAT4) is believed to stabilize interactions between two phosphorylated STAT4 dimers to form STAT4 tetramers. Here, we show that nonphosphorylated STAT4 dimers form in vivo before cytokine receptor-driven activation. Mutations in the N-domain dimerization interface abolished assembly of nonphosphorylated STAT4 dimers and prevented STAT4 phosphorylation mediated by cytokine receptors. In addition, N-domain dimerization occurred for other STAT family members but was homotypic in character. This implies a conserved role for N-domain dimerization, which might include influencing interactions with cytokine receptors, favoring homodimer formation or accelerating formation of the phosphorylated STAT dimer.

The M3 protein encoded by murine gamma herpesvirus68 (gamma HV68) functions as an immune system saboteur by the engagement of chemoattractant cytokines, thereby altering host antiviral inflammatory responses. Here we report the crystal structures of M3 both alone and in complex with the CC chemokine MCP-1. M3 is a two-domain beta sandwich protein with a unique sequence and topology, forming a tightly packed anti-parallel dimer. The stoichiometry of the MCP-1:M3 complex is 2:2, with two monomeric chemokines embedded at distal ends of the preassociated M3 dimer. Conformational flexibility and electrostatic complementation are both used by M3 to achieve high-affinity and broad-spectrum chemokine engagement. M3 also employs structural mimicry to promiscuously sequester chemokines, engaging conservative structural elements associated with both chemokine homodimerization and binding to G protein-coupled receptors.

Clathrin-mediated endocytosis depends upon the interaction of accessory proteins with the alpha-ear of the AP-2 adaptor. We present structural characterization of these regulatory interactions. DPF and DPW motif peptides derived from eps15 and epsin bind in type I beta turn conformations to a conserved pocket on the alpha-ear platform. We show evidence for a second binding site that is DPW motif specific. The structure of a complex with an AP-2 binding segment from amphiphysin reveals a novel binding motif that we term FxDxF, which is engaged in an extended conformation by a unique surface of the platform domain. The FxDxF motif is also used by AP180 and the 170 kDa isoform of synaptojanin and can be found in several potential endocytic proteins, including HIP1, CD2AP, and PLAP. A mechanism of clathrin assembly regulation is suggested by three different AP-2 engagement modes.

In a systematic study of molecular recognition of amino acid derivatives in solid-state beta-cyclodextrin (beta-CD) complexes, we have determined crystal structures for complexes of beta-cyclodextrin/N-acetyl-L-phenylalanine at 298 and 20 K and for N-acetyl-D-phenylalanine at 298 K. The crystal structures for the N-acetyl-L-phenylalanine complex present disordered inclusion complexes for which the distribution of guest molecules at room temperature is not resolvable; however, they can be located with considerable confidence at low temperature. In contrast, the complex with N-acetyl-D-phenylalanine is well ordered at room temperature. The latter complex presents an example of a complex in this series in which a water molecule is included deeply in the hydrophobic torus of the extended dimer host. In an effort to understand the mechanisms of molecular recognition giving rise to the dramatic differences in crystallographic order in these crystal structures, we have examined the intermolecular interactions in detail and have examined insertion of the enantiomer of the D-complex into the chiral beta-CD complex crystal lattice.

Clathrin-mediated endocytosis is a major pathway for the internalization of macromolecules into the cytoplasm of eukaryotic cells. The principle coat components, clathrin and the AP-2 adaptor complex, assemble a polyhedral lattice at plasma membrane bud sites with the aid of several endocytic accessory proteins. Here, we show that huntingtin-interacting protein 1 (HIP1), a binding partner of huntingtin, copurifies with brain clathrin-coated vesicles and associates directly with both AP-2 and clathrin. The discrete interaction sequences within HIP1 that facilitate binding are analogous to motifs present in other accessory proteins, including AP180, amphiphysin, and epsin. Bound to a phosphoinositide-containing membrane surface via an epsin N-terminal homology (ENTH) domain, HIP1 associates with AP-2 to provide coincident clathrin-binding sites that together efficiently recruit clathrin to the bilayer. Our data implicate HIP1 in endocytosis, and the similar modular architecture and function of HIP1, epsin, and AP180 suggest a common role in lipid-regulated clathrin lattice biogenesis.