RESUMO
Hemostasis relies on a reaction network of serine proteases and their cofactors to form a blood clot. Coagulation factor IXa (protease) plays an essential role in hemostasis as evident from the bleeding disease associated with its absence. RNA aptamers specifically targeting individual coagulation factors have potential as anticoagulants and as probes of the relationship between structure and function. Here, we report X-ray structures of human factor IXa without a ligand bound to the active site either in the apo-form or in complex with an inhibitory aptamer specific for factor IXa. The aptamer binds to an exosite in the catalytic domain and allosterically distorts the active site. Our studies reveal a conformational ensemble of IXa states, wherein large movements of Trp215 near the active site drive functional transitions between the closed (aptamer-bound), latent (apo), and open (substrate-bound) states. The latent state of the apo-enzyme may bear on the uniquely poor catalytic activity of IXa compared to other coagulation proteases. The exosite, to which the aptamer binds, has been implicated in binding VIIIa and heparin, both of which regulate IXa function. Our findings reveal the importance of exosite-driven allosteric modulation of IXa function and new strategies to rebalance hemostasis for therapeutic gain.
Assuntos
Aptâmeros de Nucleotídeos , Fator IXa , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Fator IXa/metabolismo , Fator IXa/química , Fator IXa/antagonistas & inibidores , Humanos , Regulação Alostérica , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Anticoagulantes/química , Anticoagulantes/metabolismo , Anticoagulantes/farmacologiaRESUMO
BACKGROUND: Mucin 16 (MUC16) overexpression is linked with cancer progression, metastasis, and therapy resistance in high grade serous ovarian cancer and other malignancies. The cleavage of MUC16 forms independent bimodular fragments, the shed tandem repeat sequence which circulates as a protein bearing the ovarian cancer biomarker (CA125) and a proximal membrane-bound component which is critical in MUC16 oncogenic behavior. A humanized, high affinity antibody targeting the proximal ectodomain represents a potential therapeutic agent against MUC16 with lower antigenic potential and restricted human tissue expression. RESULTS: Here, we demonstrate the potential therapeutic versatility of the humanized antibody as a monoclonal antibody, antibody drug conjugate, and chimeric antigen receptor. We report the crystal structures of 4H11-scFv, derived from an antibody specifically targeting the MUC16 C-terminal region, alone and in complex with a 26-amino acid MUC16 segment resolved at 2.36 Å and 2.47 Å resolution, respectively. The scFv forms a robust interaction with an epitope consisting of two consecutive ß-turns and a ß-hairpin stabilized by 2 hydrogen bonds. The VH-VL interface within the 4H11-scFv is stabilized through an intricate network of 11 hydrogen bonds and a cation-π interaction. CONCLUSIONS: Together, our studies offer insight into antibody-MUC16 ectodomain interaction and advance our ability to design agents with potentially improved therapeutic properties over anti-CA125 moiety antibodies.
Assuntos
Reações Antígeno-Anticorpo , Antígeno Ca-125 , Proteínas de Membrana , Feminino , Humanos , Antígeno Ca-125/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias Ovarianas/patologiaRESUMO
6-Hydroxynicotinic acid 3-monooxygenase (NicC) is a bacterial enzyme involved in the degradation of nicotinic acid. This enzyme is a Class A flavin-dependent monooxygenase that catalyzes a unique decarboxylative hydroxylation. The unliganded structure of this enzyme has previously been reported and studied using steady- and transient-state kinetics to support a comprehensive kinetic mechanism. Here we report the crystal structure of the H47Q NicC variant in both a ligand-bound (solved to 2.17 Å resolution) and unliganded (1.51 Å resolution) form. Interestingly, in the liganded form, H47Q NicC is bound to 2-mercaptopyridine (2-MP), a contaminant present in the commercial stock of 6-mercaptopyridine-3-carboxylic acid(6-MNA), a substrate analogue. 2-MP binds weakly to H47Q NicC and is not a substrate for the enzyme. Based on kinetic and thermodynamic characterization, we have fortuitously captured a catalytically inactive H47Q NicCâ¢2-MP complex in our crystal structure. This complex reveals interesting mechanistic details about the reaction catalyzed by 6-hydroxynicotinic acid 3-monooxygenase.
Assuntos
Flavina-Adenina Dinucleotídeo , Oxigenases de Função Mista , Ligantes , Flavina-Adenina Dinucleotídeo/química , Oxigenases de Função Mista/química , CinéticaRESUMO
Botulinum neurotoxin E (BoNT/E) is one of the major causes of human botulism and paradoxically also a promising therapeutic agent. Here we determined the co-crystal structures of the receptor-binding domain of BoNT/E (HCE) in complex with its neuronal receptor synaptic vesicle glycoprotein 2A (SV2A) and a nanobody that serves as a ganglioside surrogate. These structures reveal that the protein-protein interactions between HCE and SV2 provide the crucial location and specificity information for HCE to recognize SV2A and SV2B, but not the closely related SV2C. At the same time, HCE exploits a separated sialic acid-binding pocket to mediate recognition of an N-glycan of SV2. Structure-based mutagenesis and functional studies demonstrate that both the protein-protein and protein-glycan associations are essential for SV2A-mediated cell entry of BoNT/E and for its potent neurotoxicity. Our studies establish the structural basis to understand the receptor-specificity of BoNT/E and to engineer BoNT/E variants for new clinical applications.
Assuntos
Toxinas Botulínicas Tipo A , Vesículas Sinápticas , Humanos , Vesículas Sinápticas/metabolismo , Toxinas Botulínicas Tipo A/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ligação ProteicaRESUMO
Botulinum neurotoxins (BoNTs) are among the most lethal toxins known to humans, comprising seven established serotypes termed BoNT/A-G encoded in two types of gene clusters (ha and orfX) in BoNT-producing clostridia. The ha cluster encodes four non-toxic neurotoxin-associated proteins (NAPs) that assemble with BoNTs to protect and enhance their oral toxicity. However, the structure and function of the orfX-type NAPs remain largely unknown. Here, we report the crystal structures for OrfX1, OrfX2, and an OrfX1-OrfX3 complex, which are encoded in the orfX cluster of a BoNT/E1-producing Clostridium botulinum strain associated with human foodborne botulism. These structures lay the foundation for future studies on the potential roles of OrfX proteins in oral intoxication and pathogenesis of BoNTs.
Assuntos
Toxinas Botulínicas Tipo A , Clostridium botulinum , Humanos , Clostridium botulinum/genética , Clostridium botulinum/química , Clostridium botulinum/metabolismo , Toxinas Botulínicas Tipo A/metabolismo , Família MultigênicaRESUMO
Toxin A (TcdA) and toxin B (TcdB) are two key virulence factors secreted by Clostridioides difficile, which is listed as an urgent threat by the CDC. These two large homologous exotoxins are mainly responsible for diseases associated with C. difficile infection (CDI) with symptoms ranging from diarrhea to life threatening pseudomembranous colitis. Single-domain camelid antibodies (VHHs) AH3 and AA6 are two potent antitoxins against TcdA, which when combined with two TcdB-targeting VHHs showed effective protection against both primary and recurrent CDI in animal models. Here, we report the co-crystal structures of AH3 and AA6 when they form complexes with the glucosyltransferase domain (GTD) and a fragment of the delivery and receptor-binding domain (DRBD) of TcdA, respectively. Based on these structures, we find that AH3 binding enhances the overall stability of the GTD and interferes with its unfolding at acidic pH, and AA6 may inhibit the pH-dependent conformational changes in the DRBD that is necessary for pore formation of TcdA. These studies reveal two functionally critical epitopes on TcdA and shed new insights into neutralizing mechanisms and potential development of epitope-focused vaccines against TcdA.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Anticorpos de Domínio Único , Animais , EpitoposRESUMO
Background: This paper presents a review of the literature and trends related to social values and sustainable development and describes a set of case studies from a variety of community-based projects which illustrate the advantages that social values bring about as part of efforts to promote sustainability. Three approaches were used to develop this study: a bibliometric analysis of the topic "social values and sustainable development", an analysis of case studies that concretely present community projects addressing social values and sustainability, and the development of a framework linking up bibliometric clusters and the cases studies. Results: While the bibliometric analysis revealed clusters where social values are strongly connected with sustainable development, the case studies indicated the lack of a common terminology and understanding of the relation between social values, sustainable development, and community-based projects. Conclusions: The study concludes by suggesting a set of measures that could be deployed to better take social values into account when planning policies or making decisions related to community projects.
RESUMO
Clostridioides difficile is one of the most common causes of antibiotic-associated diarrhea in developed countries. As key virulence factors of C. difficile, toxin A (TcdA) and toxin B (TcdB) act by glucosylating and inactivating Rho and Ras family small GTPases in host cells, which leads to actin cytoskeleton disruption, cell rounding, and ultimately cell death. Here we present the co-crystal structure of the glucosyltransferase domain (GTD) of TcdA in complex with its substrate human RhoA at 2.60-angstrom resolution. This structure reveals that TcdA GTD grips RhoA mainly through its switch I and switch II regions, which is complemented by interactions involving RhoA's pre-switch I region. Comprehensive structural comparisons between the TcdA GTD-RhoA complex and the structures of TcdB GTD in complex with Cdc42 and R-Ras reveal both the conserved and divergent features of these two toxins in terms of substrate recognition. Taken together, these findings establish the structural basis for TcdA recognition of small GTPases and advance our understanding of the substrates selectivity of large clostridial toxins.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Proteínas Monoméricas de Ligação ao GTP , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Enterotoxinas/metabolismo , Glucosiltransferases/metabolismo , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Clostridioides difficile toxin A and B (TcdA and TcdB) are two major virulence factors responsible for diseases associated with C. difficile infection (CDI). Here, we report the 3.18-Å resolution crystal structure of a TcdA fragment (residues L843-T2481), which advances our understanding of the complete structure of TcdA holotoxin. Our structural analysis, together with complementary single molecule FRET and limited proteolysis studies, reveal that TcdA adopts a dynamic structure and its CROPs domain can sample a spectrum of open and closed conformations in a pH-dependent manner. Furthermore, a small globular subdomain (SGS) and the CROPs protect the pore-forming region of TcdA in the closed state at neutral pH, which could contribute to modulating the pH-dependent pore formation of TcdA. A rationally designed TcdA mutation that trapped the CROPs in the closed conformation showed drastically reduced cytotoxicity. Taken together, these studies shed new lights into the conformational dynamics of TcdA and its roles in TcdA intoxication.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Conformação MolecularRESUMO
Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.
Assuntos
Toxinas Botulínicas/antagonistas & inibidores , Peptídeo Hidrolases/química , Anticorpos de Domínio Único , Animais , Toxinas Botulínicas/química , Inibidores de Proteases/farmacologia , Domínios Proteicos/efeitos dos fármacos , Anticorpos de Domínio Único/farmacologia , Relação Estrutura-AtividadeRESUMO
WNTs play key roles in development and disease, signaling through Frizzled (FZD) seven-pass transmembrane receptors and numerous co-receptors including ROR and RYK family receptor tyrosine kinases (RTKs). We describe crystal structures and WNT-binding characteristics of extracellular regions from the Drosophila ROR and RYK orthologs Nrk (neurospecific receptor tyrosine kinase) and Derailed-2 (Drl-2), which bind WNTs though a FZD-related cysteine-rich domain (CRD) and WNT-inhibitory factor (WIF) domain respectively. Our crystal structures suggest that neither Nrk nor Drl-2 can accommodate the acyl chain typically attached to WNTs. The Nrk CRD contains a deeply buried bound fatty acid, unlikely to be exchangeable. The Drl-2 WIF domain lacks the lipid-binding site seen in WIF-1. We also find that recombinant DWnt-5 can bind Drosophila ROR and RYK orthologs despite lacking an acyl chain. Alongside analyses of WNT/receptor interaction sites, our structures provide further insight into how WNTs may recruit RTK co-receptors into signaling complexes.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Modelos Moleculares , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Células Sf9 , Relação Estrutura-Atividade , Proteínas Wnt/genéticaRESUMO
Toxin B (TcdB) is a primary cause of Clostridioides difficile infection (CDI). This toxin acts by glucosylating small GTPases in the Rho/Ras families, but the structural basis for TcdB recognition and selectivity of specific GTPase substrates remain unsolved. Here, we report the cocrystal structures of the glucosyltransferase domain (GTD) of two distinct TcdB variants in complex with human Cdc42 and R-Ras, respectively. These structures reveal a common structural mechanism by which TcdB recognizes Rho and R-Ras. Furthermore, we find selective clustering of adaptive residue changes in GTDs that determine their substrate preferences, which helps partition all known TcdB variants into two groups that display distinct specificities toward Rho or R-Ras. Mutations that selectively disrupt GTPases binding reduce the glucosyltransferase activity of the GTD and the toxicity of TcdB holotoxin. These findings establish the structural basis for TcdB recognition of small GTPases and reveal strategies for therapeutic interventions for CDI.
RESUMO
Botulinum neurotoxin serotype E (BoNT/E) is one of the major causes of human botulism, which is a life-threatening disease caused by flaccid paralysis of muscles. After receptor-mediated toxin internalization into motor neurons, the translocation domain (HN) of BoNT/E transforms into a protein channel upon vesicle acidification in endosomes and delivers its protease domain (LC) across membrane to enter the neuronal cytosol. It is believed that the rapid onset of BoNT/E intoxication compared to other BoNT serotypes is related to its swift internalization and translocation. We recently identified two neutralizing single-domain camelid antibodies (VHHs) against BoNT/E1 termed JLE-E5 and JLE-E9. Here, we report the crystal structures of these two VHHs bound to the LCHN domain of BoNT/E1. The structures reveal that these VHHs recognize two distinct epitopes that are partially overlapping with the putative transmembrane regions on HN, and therefore could physically block membrane association of BoNT/E1. This is confirmed by our in vitro studies, which show that these VHHs inhibit the structural change of BoNT/E1 at acidic pH and interfere with BoNT/E1 association with lipid vesicles. Therefore, these two VHHs neutralize BoNT/E1 by preventing the transmembrane delivery of LC. Furthermore, structure-based sequence analyses show that the 3-dimensional epitopes of these two VHHs are largely conserved across many BoNT/E subtypes, suggesting a broad-spectrum protection against the BoNT/E family. In summary, this work improves our understanding of the membrane translocation mechanism of BoNT/E and paves the way for developing VHHs as diagnostics or therapeutics for the treatment of BoNT/E intoxication.
Assuntos
Anticorpos Neutralizantes/farmacologia , Toxinas Botulínicas/antagonistas & inibidores , Membrana Celular/efeitos dos fármacos , Membranas Artificiais , Anticorpos de Domínio Único/farmacologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos , Transporte Biológico , Toxinas Botulínicas/genética , Toxinas Botulínicas/imunologia , Toxinas Botulínicas/metabolismo , Membrana Celular/metabolismo , Epitopos , Interações Hospedeiro-Patógeno , Mutação , Conformação Proteica , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Relação Estrutura-AtividadeRESUMO
Botulinum neurotoxin (BoNT) is one of the most acutely lethal toxins known to humans, and effective treatment for BoNT intoxication is urgently needed. Single-domain antibodies (VHH) have been examined as a countermeasure for BoNT because of their high stability and ease of production. Here, we investigate the structures and the neutralization mechanisms for six unique VHHs targeting BoNT/A1 or BoNT/B1. These studies reveal diverse neutralizing mechanisms by which VHHs prevent host receptor binding or block transmembrane delivery of the BoNT protease domain. Guided by this knowledge, we design heterodimeric VHHs by connecting two neutralizing VHHs via a flexible spacer so they can bind simultaneously to the toxin. These bifunctional VHHs display much greater potency in a mouse co-intoxication model than similar heterodimers unable to bind simultaneously. Taken together, our studies offer insight into antibody neutralization of BoNTs and advance our ability to design multivalent anti-pathogen VHHs with improved therapeutic properties.
Assuntos
Antitoxinas/química , Toxinas Botulínicas/antagonistas & inibidores , Desenho de Fármacos , Anticorpos de Domínio Único/química , Animais , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Toxinas Botulínicas/química , Membrana Celular/metabolismo , Feminino , Concentração de Íons de Hidrogênio , Camundongos , Modelos Moleculares , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Receptores de Superfície Celular/metabolismoRESUMO
Clostridium difficile is an opportunistic pathogen that establishes in the colon when the gut microbiota are disrupted by antibiotics or disease. C. difficile infection (CDI) is largely caused by two virulence factors, TcdA and TcdB. Here, we report a 3.87-Å-resolution crystal structure of TcdB holotoxin that captures a unique conformation of TcdB at endosomal pH. Complementary biophysical studies suggest that the C-terminal combined repetitive oligopeptides (CROPs) domain of TcdB is dynamic and can sample open and closed conformations that may facilitate modulation of TcdB activity in response to environmental and cellular cues during intoxication. Furthermore, we report three crystal structures of TcdB-antibody complexes that reveal how antibodies could specifically inhibit the activities of individual TcdB domains. Our studies provide novel insight into the structure and function of TcdB holotoxin and identify intrinsic vulnerabilities that could be exploited to develop new therapeutics and vaccines for the treatment of CDI.
Assuntos
Proteínas de Bactérias/química , Toxinas Bacterianas/química , Clostridioides difficile/química , Sequência de Aminoácidos , Anticorpos Neutralizantes/imunologia , Complexo Antígeno-Anticorpo/química , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Sequência Conservada , Cristalografia por Raios X , Endossomos/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Lipossomos , Potenciais da Membrana , Modelos Moleculares , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Glucokinase from pathogenic protozoa of the genus Leishmania is a potential drug target for the chemotherapeutic treatment against leishmaniasis because this enzyme is located at a nodal point between two critically important metabolic pathways, glycolysis and the pentose phosphate pathway (PPP). L. braziliensis glucokinase (LbGlcK) was evaluated for its structural characterization and enzymatic performance. The enzyme catalyzes the phosphorylation of d-glucose with co-substrate ATP to yield the products G6P and ADP. LbGlcK had KM values determined as 6.61 ± 2.63 mM and 0.338 ± 0.080 mM for d-glucose and ATP, respectively. The 1.85 Å resolution X-ray crystal structure of the apo form of LbGlcK was determined and a homodimer was revealed where each subunit (both in open conformations) included the typical small and large domains. Structural comparisons were assessed in relationship to Homo sapiens hexokinase IV and Trypanosoma cruzi glucokinase. Comparisons revealed that all residues important for making hydrogen bonding interactions with d-glucose in the active site and catalysis were strictly conserved. LbGlcK was screened against four glucosamine analogue inhibitors and the stronger inhibitor of the series, HPOP-GlcN, had a Ki value of 56.9 ± 16.6 µM that exhibited competitive inhibition. For the purpose of future structure-based drug design experimentation, L. braziliensis glucokinase was observed to be very similar to T. cruzi glucokinase even though there was a 44% protein sequence identity between the two enzymes.
Assuntos
Glucoquinase/química , Glucoquinase/metabolismo , Leishmania braziliensis/enzimologia , Leishmaniose Cutânea/parasitologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Glucoquinase/genética , Glucose/metabolismo , Humanos , Cinética , Leishmania braziliensis/química , Leishmania braziliensis/genética , Modelos Moleculares , Fosforilação , Proteínas de Protozoários/genética , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Botulinum neurotoxin (BoNT) delivers its protease domain across the vesicle membrane to enter the neuronal cytosol upon vesicle acidification. This process is mediated by its translocation domain (HN), but the molecular mechanism underlying membrane insertion of HN remains poorly understood. Here, we report two crystal structures of BoNT/A1 HN that reveal a novel molecular switch (termed BoNT-switch) in HN, where buried α-helices transform into surface-exposed hydrophobic ß-hairpins triggered by acidic pH. Locking the BoNT-switch by disulfide trapping inhibited the association of HN with anionic liposomes, blocked channel formation by HN, and reduced the neurotoxicity of BoNT/A1 by up to ~180-fold. Single particle counting studies showed that an acidic environment tends to promote BoNT/A1 self-association on liposomes, which is partly regulated by the BoNT-switch. These findings suggest that the BoNT-switch flips out upon exposure to the acidic endosomal pH, which enables membrane insertion of HN that subsequently leads to LC delivery.
Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Membranas Intracelulares/metabolismo , Neurônios/metabolismo , Neurotoxinas/metabolismo , Sequência de Aminoácidos , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/isolamento & purificação , Cristalografia por Raios X , Citosol/metabolismo , Endossomos/metabolismo , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Lipossomos/metabolismo , Modelos Moleculares , Neurônios/citologia , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas do Envelope Viral/químicaRESUMO
Mitochondrial calcium uptake is critical for regulating ATP production, intracellular calcium signalling, and cell death. This uptake is mediated by a highly selective calcium channel called the mitochondrial calcium uniporter (MCU). Here, we determined the structures of the pore-forming MCU proteins from two fungi by X-ray crystallography and single-particle cryo-electron microscopy. The stoichiometry, overall architecture, and individual subunit structure differed markedly from those described in the recent nuclear magnetic resonance structure of Caenorhabditis elegans MCU. We observed a dimer-of-dimer architecture across species and chemical environments, which was corroborated by biochemical experiments. Structural analyses and functional characterization uncovered the roles of key residues in the pore. These results reveal a new ion channel architecture, provide insights into calcium coordination, selectivity and conduction, and establish a structural framework for understanding the mechanism of mitochondrial calcium uniporter function.
Assuntos
Canais de Cálcio/química , Canais de Cálcio/ultraestrutura , Microscopia Crioeletrônica , Fusarium/química , Metarhizium/química , Animais , Caenorhabditis elegans/química , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Cristalografia por Raios X , Ativação do Canal Iônico , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Reprodutibilidade dos Testes , SolubilidadeRESUMO
Unfractionated heparin (UFH), the standard anticoagulant for cardiopulmonary bypass (CPB) surgery, carries a risk of post-operative bleeding and is potentially harmful in patients with heparin-induced thrombocytopenia-associated antibodies. To improve the activity of an alternative anticoagulant, the RNA aptamer 11F7t, we solved X-ray crystal structures of the aptamer bound to factor Xa (FXa). The finding that 11F7t did not bind the catalytic site suggested that it could complement small-molecule FXa inhibitors. We demonstrate that combinations of 11F7t and catalytic-site FXa inhibitors enhance anticoagulation in purified reaction mixtures and plasma. Aptamer-drug combinations prevented clot formation as effectively as UFH in human blood circulated in an extracorporeal oxygenator circuit that mimicked CPB, while avoiding side effects of UFH. An antidote could promptly neutralize the anticoagulant effects of both FXa inhibitors. Our results suggest that drugs and aptamers with shared targets can be combined to exert more specific and potent effects than either agent alone.
Assuntos
Anticoagulantes/administração & dosagem , Inibidores do Fator Xa/administração & dosagem , Fator Xa/química , Hemorragia Pós-Operatória/tratamento farmacológico , Anticoagulantes/química , Aptâmeros de Nucleotídeos/administração & dosagem , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Ponte Cardiopulmonar/efeitos adversos , Cristalografia por Raios X , Combinação de Medicamentos , Fator Xa/genética , Inibidores do Fator Xa/química , Heparina/efeitos adversos , Humanos , Hemorragia Pós-Operatória/genética , Hemorragia Pós-Operatória/patologia , Conformação Proteica/efeitos dos fármacosRESUMO
Clostridium difficile infection is the most common cause of antibiotic-associated diarrhea in developed countries. The major virulence factor, C. difficile toxin B (TcdB), targets colonic epithelia by binding to the frizzled (FZD) family of Wnt receptors, but how TcdB recognizes FZDs is unclear. Here, we present the crystal structure of a TcdB fragment in complex with the cysteine-rich domain of human FZD2 at 2.5-angstrom resolution, which reveals an endogenous FZD-bound fatty acid acting as a co-receptor for TcdB binding. This lipid occupies the binding site for Wnt-adducted palmitoleic acid in FZDs. TcdB binding locks the lipid in place, preventing Wnt from engaging FZDs and signaling. Our findings establish a central role of fatty acids in FZD-mediated TcdB pathogenesis and suggest strategies to modulate Wnt signaling.