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1.
Nat Rev Microbiol ; 19(11): 685-700, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34535791

RESUMO

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an unprecedented global health crisis. However, therapeutic options for treatment are still very limited. The development of drugs that target vital proteins in the viral life cycle is a feasible approach for treating COVID-19. Belonging to the subfamily Orthocoronavirinae with the largest RNA genome, SARS-CoV-2 encodes a total of 29 proteins. These non-structural, structural and accessory proteins participate in entry into host cells, genome replication and transcription, and viral assembly and release. SARS-CoV-2 proteins can individually perform essential physiological roles, be components of the viral replication machinery or interact with numerous host cellular factors. In this Review, we delineate the structural features of SARS-CoV-2 from the whole viral particle to the individual viral proteins and discuss their functions as well as their potential as targets for therapeutic interventions.

2.
Nat Commun ; 12(1): 4621, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330928

RESUMO

Cytochromes bd are ubiquitous amongst prokaryotes including many human-pathogenic bacteria. Such complexes are targets for the development of antimicrobial drugs. However, an understanding of the relationship between the structure and functional mechanisms of these oxidases is incomplete. Here, we have determined the 2.8 Å structure of Mycobacterium smegmatis cytochrome bd by single-particle cryo-electron microscopy. This bd oxidase consists of two subunits CydA and CydB, that adopt a pseudo two-fold symmetrical arrangement. The structural topology of its Q-loop domain, whose function is to bind the substrate, quinol, is significantly different compared to the C-terminal region reported for cytochromes bd from Geobacillus thermodenitrificans (G. th) and Escherichia coli (E. coli). In addition, we have identified two potential oxygen access channels in the structure and shown that similar tunnels also exist in G. th and E. coli cytochromes bd. This study provides insights to develop a framework for the rational design of antituberculosis compounds that block the oxygen access channels of this oxidase.


Assuntos
Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica/métodos , Grupo dos Citocromos b/ultraestrutura , Complexo de Proteínas da Cadeia de Transporte de Elétrons/ultraestrutura , Mycobacterium smegmatis/enzimologia , Oxirredutases/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Heme/química , Heme/metabolismo , Modelos Moleculares , Mycobacterium smegmatis/genética , Oxirredutases/química , Oxirredutases/metabolismo , Oxigênio/metabolismo , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato
3.
Nat Commun ; 12(1): 4134, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226547

RESUMO

Junin virus (JUNV) causes Argentine hemorrhagic fever, a debilitating human disease of high mortality rates and a great risk to public health worldwide. Studying the L protein that replicates and transcribes the genome of JUNV, and its regulator Z protein should provide critical clues to identify therapeutic targets for disrupting the life cycle of JUNV. Here we report the 3.54 Å cryo-EM structure of the JUNV L protein complexed with regulator Z protein. JUNV L structure reveals a conserved architecture containing signature motifs found in other L proteins. Structural analysis shows that L protein is regulated by binding of Z protein at the RNA product exit site. Based on these findings, we propose a model for the role of Z protein as a switch to turn on/off the viral RNA synthesis via its interaction with L protein. Our work unveils the mechanism of JUNV transcription, replication and regulation, which provides a framework for the rational design of antivirals for combating viral infections.


Assuntos
Arenavirus/enzimologia , Arenavirus/genética , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Microscopia Crioeletrônica , Febre Hemorrágica Americana/virologia , Interações Hospedeiro-Patógeno , Humanos , Vírus Junin/enzimologia , Vírus Junin/genética , Modelos Moleculares , Conformação Proteica , RNA Viral
4.
Cell ; 184(13): 3474-3485.e11, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34143953

RESUMO

The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (7MeGpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.


Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus/genética , Exorribonucleases/genética , Metiltransferases/genética , SARS-CoV-2/genética , Sequência de Aminoácidos , COVID-19/virologia , Humanos , RNA Mensageiro/genética , RNA Viral/genética , Alinhamento de Sequência , Transcrição Genética/genética , Replicação Viral/genética
5.
Nat Commun ; 12(1): 3061, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031399

RESUMO

The SARS-CoV-2 pandemic has triggered global efforts to develop therapeutics. The main protease of SARS-CoV-2 (Mpro), critical for viral replication, is a key target for therapeutic development. An organoselenium drug called ebselen has been demonstrated to have potent Mpro inhibition and antiviral activity. We have examined the binding modes of ebselen and its derivative in Mpro via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger Mpro inhibition than ebselen and potent ability to rescue infected cells were observed for a number of derivatives. A free selenium atom bound with cysteine of catalytic dyad has been revealed in crystallographic structures of Mpro with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct and formation of a phenolic by-product, confirmed by mass spectrometry. The target engagement with selenation mechanism of inhibition suggests wider therapeutic applications of these compounds against SARS-CoV-2 and other zoonotic beta-corona viruses.


Assuntos
Azóis/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Compostos Organosselênicos/farmacologia , SARS-CoV-2/enzimologia , Antivirais/farmacologia , Azóis/química , Domínio Catalítico , Proteases 3C de Coronavírus/metabolismo , Cristalografia por Raios X , Cisteína/química , Hidrólise , Modelos Moleculares , Compostos Organosselênicos/química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Padrões de Referência , SARS-CoV-2/efeitos dos fármacos , Salicilanilidas/química , Salicilanilidas/farmacologia , Selênio/metabolismo
6.
Nat Commun ; 12(1): 2623, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976198

RESUMO

COVID-19 pandemic caused by SARS-CoV-2 constitutes a global public health crisis with enormous economic consequences. Monoclonal antibodies against SARS-CoV-2 can provide an important treatment option to fight COVID-19, especially for the most vulnerable populations. In this work, potent antibodies binding to SARS-CoV-2 Spike protein were identified from COVID-19 convalescent patients. Among them, P4A1 interacts directly with and covers majority of the Receptor Binding Motif of the Spike Receptor-Binding Domain, shown by high-resolution complex structure analysis. We further demonstrate the binding and neutralizing activities of P4A1 against wild type and mutant Spike proteins or pseudoviruses. P4A1 was subsequently engineered to reduce the potential risk for Antibody-Dependent Enhancement of infection and to extend its half-life. The engineered antibody exhibits an optimized pharmacokinetic and safety profile, and it results in complete viral clearance in a rhesus monkey model of COVID-19 following a single injection. These data suggest its potential against SARS-CoV-2 related diseases.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Especificidade de Anticorpos/imunologia , COVID-19/tratamento farmacológico , COVID-19/epidemiologia , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Feminino , Humanos , Macaca mulatta , Masculino , Mutação , Pandemias , Ligação Proteica , Domínios Proteicos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Resultado do Tratamento , Células Vero
7.
PLoS Biol ; 19(5): e3001209, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33961621

RESUMO

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) threatens global public health and economy unprecedentedly, requiring accelerating development of prophylactic and therapeutic interventions. Molecular understanding of neutralizing antibodies (NAbs) would greatly help advance the development of monoclonal antibody (mAb) therapy, as well as the design of next generation recombinant vaccines. Here, we applied H2L2 transgenic mice encoding the human immunoglobulin variable regions, together with a state-of-the-art antibody discovery platform to immunize and isolate NAbs. From a large panel of isolated antibodies, 25 antibodies showed potent neutralizing activities at sub-nanomolar levels by engaging the spike receptor-binding domain (RBD). Importantly, one human NAb, termed PR1077, from the H2L2 platform and 2 humanized NAb, including PR953 and PR961, were further characterized and subjected for subsequent structural analysis. High-resolution X-ray crystallography structures unveiled novel epitopes on the receptor-binding motif (RBM) for PR1077 and PR953, which directly compete with human angiotensin-converting enzyme 2 (hACE2) for binding, and a novel non-blocking epitope on the neighboring site near RBM for PR961. Moreover, we further tested the antiviral efficiency of PR1077 in the Ad5-hACE2 transduction mouse model of COVID-19. A single injection provided potent protection against SARS-CoV-2 infection in either prophylactic or treatment groups. Taken together, these results shed light on the development of mAb-related therapeutic interventions for COVID-19.


Assuntos
Anticorpos Neutralizantes/imunologia , COVID-19/virologia , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/imunologia , COVID-19/epidemiologia , COVID-19/imunologia , COVID-19/metabolismo , Epitopos/imunologia , Humanos , Camundongos , Camundongos Transgênicos , Testes de Neutralização , Pandemias , Ligação Proteica , Domínios Proteicos , Receptores Virais/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia
8.
PLoS Pathog ; 17(4): e1009507, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33909694

RESUMO

The development of a universal vaccine against foot-and-mouth disease virus (FMDV) is hindered by cross-serotype antigenic diversity and by a lack of knowledge regarding neutralization of the virus in natural hosts. In this study, we isolated serotype O-specific neutralizing antibodies (NAbs) (F145 and B77) from recovered natural bovine hosts by using the single B cell antibody isolation technique. We also identified a serotype O/A cross-reacting NAb (R50) and determined virus-NAb complex structures by cryo-electron microscopy at near-atomic resolution. F145 and B77 were shown to engage the capsid of FMDV-O near the icosahedral threefold axis, binding to the BC/HI-loop of VP2. In contrast, R50 engages the capsids of both FMDV-O and FMDV-A between the 2- and 5-fold axes and binds to the BC/EF/GH-loop of VP1 and to the GH-loop of VP3 from two adjacent protomers, revealing a previously unknown antigenic site. The cross-serotype neutralizing epitope recognized by R50 is highly conserved among serotype O/A. These findings help to elucidate FMDV neutralization by natural hosts and provide epitope information for the development of a universal vaccine for cross-serotype protection against FMDV.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Febre Aftosa/imunologia , Febre Aftosa/virologia , Animais , Variação Antigênica , Capsídeo/imunologia , Bovinos , Microscopia Crioeletrônica/veterinária , Epitopos/imunologia , Vírus da Febre Aftosa/ultraestrutura , Sorogrupo
10.
Proc Natl Acad Sci U S A ; 118(16)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33853951

RESUMO

Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is known about how they interact and function. Here, we have isolated a native cargo-packaging encapsulin from Mycobacterium smegmatis and determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). This encapsulin comprises an icosahedral shell and a dodecameric DyP cargo. The dodecameric DyP consists of two hexamers with a twofold axis of symmetry and stretches across the interior of the encapsulin. Our results reveal that the encapsulin shell plays a role in stabilizing the dodecameric DyP. Furthermore, we have proposed a potential mechanism for removing the hydrogen peroxide based on the structural features. Our study also suggests that the DyP is the primary cargo protein of mycobacterial encapsulins and is a potential target for antituberculosis drug discovery.

11.
Protein Cell ; 2021 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-33864621

RESUMO

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (Mpro), PLpro is responsible for processing the viral replicase polyprotein into functional units. Therefore, it is an attractive target for antiviral drug development. Here we discovered four compounds, YM155, cryptotanshinone, tanshinone I and GRL0617 that inhibit SARS-CoV-2 PLpro with IC50 values ranging from 1.39 to 5.63 µmol/L. These compounds also exhibit strong antiviral activities in cell-based assays. YM155, an anticancer drug candidate in clinical trials, has the most potent antiviral activity with an EC50 value of 170 nmol/L. In addition, we have determined the crystal structures of this enzyme and its complex with YM155, revealing a unique binding mode. YM155 simultaneously targets three "hot" spots on PLpro, including the substrate-binding pocket, the interferon stimulating gene product 15 (ISG15) binding site and zinc finger motif. Our results demonstrate the efficacy of this screening and repurposing strategy, which has led to the discovery of new drug leads with clinical potential for COVID-19 treatments.

12.
Proc Natl Acad Sci U S A ; 118(15)2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33876763

RESUMO

Complex II, also known as succinate dehydrogenase (SQR) or fumarate reductase (QFR), is an enzyme involved in both the Krebs cycle and oxidative phosphorylation. Mycobacterial Sdh1 has recently been identified as a new class of respiratory complex II (type F) but with an unknown electron transfer mechanism. Here, using cryoelectron microscopy, we have determined the structure of Mycobacterium smegmatis Sdh1 in the presence and absence of the substrate, ubiquinone-1, at 2.53-Å and 2.88-Å resolution, respectively. Sdh1 comprises three subunits, two that are water soluble, SdhA and SdhB, and one that is membrane spanning, SdhC. Within these subunits we identified a quinone-binding site and a rarely observed Rieske-type [2Fe-2S] cluster, the latter being embedded in the transmembrane region. A mutant, where two His ligands of the Rieske-type [2Fe-2S] were changed to alanine, abolished the quinone reduction activity of the Sdh1. Our structures allow the proposal of an electron transfer pathway that connects the substrate-binding and quinone-binding sites. Given the unique features of Sdh1 and its essential role in Mycobacteria, these structures will facilitate antituberculosis drug discovery efforts that specifically target this complex.

13.
Chem Commun (Camb) ; 57(12): 1430-1433, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33462575

RESUMO

The main viral protease (Mpro) of SARS-CoV-2 is a nucleophilic cysteine hydrolase and a current target for anti-viral chemotherapy. We describe a high-throughput solid phase extraction coupled to mass spectrometry Mpro assay. The results reveal some ß-lactams, including penicillin esters, are active site reacting Mpro inhibitors, thus highlighting the potential of acylating agents for Mpro inhibition.


Assuntos
Antivirais/farmacologia , Cisteína Endopeptidases/efeitos dos fármacos , Espectrometria de Massas/métodos , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , beta-Lactamas/farmacologia , Acilação , Antivirais/química , COVID-19/virologia , Domínio Catalítico , Ensaios de Triagem em Larga Escala , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , SARS-CoV-2/enzimologia , beta-Lactamas/química
14.
Cell ; 184(1): 184-193.e10, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33232691

RESUMO

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.


Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus/química , Microscopia Crioeletrônica , RNA Mensageiro/química , RNA Viral/química , SARS-CoV-2/química , Proteínas do Complexo da Replicase Viral/química , Sequência de Aminoácidos , Coronavirus/química , Coronavirus/classificação , Coronavirus/enzimologia , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Metiltransferases/metabolismo , Modelos Moleculares , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/enzimologia , Alinhamento de Sequência , Transcrição Genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
15.
Nat Commun ; 11(1): 5874, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33208736

RESUMO

Non-structural proteins (nsp) constitute the SARS-CoV-2 replication and transcription complex (RTC) to play a pivotal role in the virus life cycle. Here we determine the atomic structure of a SARS-CoV-2 mini RTC, assembled by viral RNA-dependent RNA polymerase (RdRp, nsp12) with a template-primer RNA, nsp7 and nsp8, and two helicase molecules (nsp13-1 and nsp13-2), by cryo-electron microscopy. Two groups of mini RTCs with different conformations of nsp13-1 are identified. In both of them, nsp13-1 stabilizes overall architecture of the mini RTC by contacting with nsp13-2, which anchors the 5'-extension of RNA template, as well as interacting with nsp7-nsp8-nsp12-RNA. Orientation shifts of nsp13-1 results in its variable interactions with other components in two forms of mini RTC. The mutations on nsp13-1:nsp12 and nsp13-1:nsp13-2 interfaces prohibit the enhancement of helicase activity achieved by mini RTCs. These results provide an insight into how helicase couples with polymerase to facilitate its function in virus replication and transcription.


Assuntos
Betacoronavirus/química , Betacoronavirus/fisiologia , Replicação Viral , Betacoronavirus/genética , Betacoronavirus/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Viral/metabolismo , SARS-CoV-2 , Relação Estrutura-Atividade , Transcrição Genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
16.
Sci Adv ; 6(44)2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33127676

RESUMO

In bacteria, adenosine 5'-triphosphate (ATP)-binding cassette (ABC) importers are essential for the uptake of nutrients including the nonreducing disaccharide trehalose, a metabolite that is crucial for the survival and virulence of several human pathogens including Mycobacterium tuberculosis SugABC is an ABC transporter that translocates trehalose from the periplasmic lipoprotein LpqY into the cytoplasm of mycobacteria. Here, we report four high-resolution cryo-electron microscopy structures of the mycobacterial LpqY-SugABC complex to reveal how it binds and passes trehalose through the membrane to the cytoplasm. A unique feature observed in this system is the initial mode of capture of the trehalose at the LpqY interface. Uptake is achieved by a pivotal rotation of LpqY relative to SugABC, moving from an open and accessible conformation to a clamped conformation upon trehalose binding. These findings enrich our understanding as to how ABC transporters facilitate substrate transport across the membrane in Gram-positive bacteria.

17.
Nat Commun ; 11(1): 4421, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887891

RESUMO

Receptor usage that determines cell tropism and drives viral classification closely correlates with the virus structure. Enterovirus B (EV-B) consists of several subgroups according to receptor usage, among which echovirus 30 (E30), a leading causative agent for human aseptic meningitis, utilizes FcRn as an uncoating receptor. However, receptors for many EVs remain unknown. Here we analyzed the atomic structures of E30 mature virion, empty- and A-particles, which reveals serotype-specific epitopes and striking conformational differences between the subgroups within EV-Bs. Of these, the VP1 BC loop markedly distinguishes E30 from other EV-Bs, indicative of a role as a structural marker for EV-B. By obtaining cryo-electron microscopy structures of E30 in complex with its receptor FcRn and CD55 and comparing its homologs, we deciphered the underlying molecular basis for receptor recognition. Together with experimentally derived viral receptor identifications, we developed a structure-based in silico algorithm to inform a rational prediction for EV receptor usage.


Assuntos
Complexo Antígeno-Anticorpo/ultraestrutura , Enterovirus Humano B/ultraestrutura , Antígenos Virais/ultraestrutura , Antígenos CD55/imunologia , Microscopia Crioeletrônica , Enterovirus Humano B/imunologia , Epitopos/ultraestrutura , Humanos , Receptores Fc/imunologia , Vírion/ultraestrutura
18.
Nat Commun ; 11(1): 4419, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887892

RESUMO

Echovirus 30 (E30), a serotype of Enterovirus B (EV-B), recently emerged as a major causative agent of aseptic meningitis worldwide. E30 is particularly devastating in the neonatal population and currently no vaccine or antiviral therapy is available. Here we characterize two highly potent E30-specific monoclonal antibodies, 6C5 and 4B10, which efficiently block binding of the virus to its attachment receptor CD55 and uncoating receptor FcRn. Combinations of 6C5 and 4B10 augment the sum of their individual anti-viral activities. High-resolution structures of E30-6C5-Fab and E30-4B10-Fab define the location and nature of epitopes targeted by the antibodies. 6C5 and 4B10 engage the capsid loci at the north rim of the canyon and in-canyon, respectively. Notably, these regions exhibit antigenic variability across EV-Bs, highlighting challenges in development of broad-spectrum antibodies. Our structures of these neutralizing antibodies of E30 are instructive for development of vaccines and therapeutics against EV-B infections.


Assuntos
Anticorpos Neutralizantes/ultraestrutura , Complexo Antígeno-Anticorpo/ultraestrutura , Proteínas do Capsídeo/imunologia , Enterovirus Humano B/imunologia , Animais , Anticorpos Monoclonais/ultraestrutura , Antígenos Virais , Antígenos CD55/imunologia , Microscopia Crioeletrônica , Epitopos/ultraestrutura , Humanos , Meningite Asséptica/virologia , Camundongos , Receptores Fc/imunologia , Sorogrupo
19.
Nat Commun ; 11(1): 4245, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843629

RESUMO

Diheme-containing succinate:menaquinone oxidoreductases (Sdh) are widespread in Gram-positive bacteria but little is known about the catalytic mechanisms they employ for succinate oxidation by menaquinone. Here, we present the 2.8 Å cryo-electron microscopy structure of a Mycobacterium smegmatis Sdh, which forms a trimer. We identified the membrane-anchored SdhF as a subunit of the complex. The 3 kDa SdhF forms a single transmembrane helix and this helix plays a role in blocking the canonically proximal quinone-binding site. We also identified two distal quinone-binding sites with bound quinones. One distal binding site is formed by neighboring subunits of the complex. Our structure further reveals the electron/proton transfer pathway for succinate oxidation by menaquinone. Moreover, this study provides further structural insights into the physiological significance of a trimeric respiratory complex II. The structure of the menaquinone binding site could provide a framework for the development of Sdh-selective anti-mycobacterial drugs.


Assuntos
Proteínas de Bactérias/química , Mycobacterium smegmatis/enzimologia , Succinato Desidrogenase/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Catálise , Microscopia Crioeletrônica , Transporte de Elétrons , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mycobacterium smegmatis/química , Oxirredução , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Succinato Desidrogenase/metabolismo , Ácido Succínico/metabolismo , Vitamina K 2/metabolismo
20.
Nature ; 586(7828): 317-321, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32640464

RESUMO

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, is a flavin adenine dinucleotide-, thiamine diphosphate- and magnesium-dependent enzyme that catalyses the first step in the biosynthesis of branched-chain amino acids1. It is the target for more than 50 commercial herbicides2. AHAS requires both catalytic and regulatory subunits for maximal activity and functionality. Here we describe structures of the hexadecameric AHAS complexes of Saccharomyces cerevisiae and dodecameric AHAS complexes of Arabidopsis thaliana. We found that the regulatory subunits of these AHAS complexes form a core to which the catalytic subunit dimers are attached, adopting the shape of a Maltese cross. The structures show how the catalytic and regulatory subunits communicate with each other to provide a pathway for activation and for feedback inhibition by branched-chain amino acids. We also show that the AHAS complex of Mycobacterium tuberculosis adopts a similar structure, thus demonstrating that the overall AHAS architecture is conserved across kingdoms.


Assuntos
Acetolactato Sintase/química , Arabidopsis/enzimologia , Saccharomyces cerevisiae/enzimologia , Acetolactato Sintase/metabolismo , Trifosfato de Adenosina/metabolismo , Aminoácidos de Cadeia Ramificada/biossíntese , Domínio Catalítico , Ativação Enzimática , Evolução Molecular , Retroalimentação Fisiológica , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mycobacterium tuberculosis/enzimologia , Ligação Proteica , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Valina/metabolismo
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