RESUMEN
The entry of coronaviruses is initiated by spike recognition of host cellular receptors, involving proteinaceous and/or glycan receptors. Recently, TMPRSS2 was identified as the proteinaceous receptor for HCoV-HKU1 alongside sialoglycan as a glycan receptor. However, the underlying mechanisms for viral entry remain unknown. Here, we investigated the HCoV-HKU1C spike in the inactive, glycan-activated, and functionally anchored states, revealing that sialoglycan binding induces a conformational change of the NTD and promotes the neighboring RBD of the spike to open for TMPRSS2 recognition, exhibiting a synergistic mechanism for the entry of HCoV-HKU1. The RBD of HCoV-HKU1 features an insertion subdomain that recognizes TMPRSS2 through three previously undiscovered interfaces. Furthermore, structural investigation of HCoV-HKU1A in combination with mutagenesis and binding assays confirms a conserved receptor recognition pattern adopted by HCoV-HKU1. These studies advance our understanding of the complex viral-host interactions during entry, laying the groundwork for developing new therapeutics against coronavirus-associated diseases.
Asunto(s)
Serina Endopeptidasas , Glicoproteína de la Espiga del Coronavirus , Internalización del Virus , Humanos , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Polisacáridos/metabolismo , Polisacáridos/química , Células HEK293 , Unión Proteica , Receptores Virales/metabolismo , Receptores Virales/química , Coronavirus/metabolismo , Modelos MolecularesRESUMEN
The persistence of the coronavirus disease 2019 (COVID-19) pandemic has resulted in increasingly disruptive impacts, and it has become the most devastating challenge to global health in a century. The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants challenges the currently available therapeutics for clinical application. Nonstructural proteins (also known as replicase proteins) with versatile biological functions play central roles in viral replication and transcription inside the host cells, and they are the most conserved target proteins among the SARS-CoV-2 variants. Specifically, they constitute the replication-transcription complexes (RTCs) dominating the synthesis of viral RNA. Knowledge of themolecular mechanisms of nonstructural proteins and their assembly into RTCs will benefit the development of antivirals targeting them against existing or potentially emerging variants. In this review, we summarize current knowledge of the structures and functions of coronavirus nonstructural proteins as well as the assembly and functions of RTCs in the life cycle of the virus.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Humanos , ARN Viral/genética , Replicación ViralRESUMEN
Decoration of cap on viral RNA plays essential roles in SARS-CoV-2 proliferation. Here, we report a mechanism for SARS-CoV-2 RNA capping and document structural details at atomic resolution. The NiRAN domain in polymerase catalyzes the covalent link of RNA 5' end to the first residue of nsp9 (termed as RNAylation), thus being an intermediate to form cap core (GpppA) with GTP catalyzed again by NiRAN. We also reveal that triphosphorylated nucleotide analog inhibitors can be bonded to nsp9 and fit into a previously unknown "Nuc-pocket" in NiRAN, thus inhibiting nsp9 RNAylation and formation of GpppA. S-loop (residues 50-KTN-52) in NiRAN presents a remarkable conformational shift observed in RTC bound with sofosbuvir monophosphate, reasoning an "induce-and-lock" mechanism to design inhibitors. These findings not only improve the understanding of SARS-CoV-2 RNA capping and the mode of action of NAIs but also provide a strategy to design antiviral drugs.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN , Antivirales/química , Nucleótidos/química , Proteínas no Estructurales Virales/metabolismoRESUMEN
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.
Asunto(s)
ARN Polimerasa Dependiente de ARN de Coronavirus/genética , Exorribonucleasas/genética , Metiltransferasas/genética , SARS-CoV-2/genética , Secuencia de Aminoácidos , COVID-19/virología , Humanos , ARN Mensajero/genética , ARN Viral/genética , Alineación de Secuencia , Transcripción Genética/genética , Replicación Viral/genéticaRESUMEN
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.
Asunto(s)
ARN Polimerasa Dependiente de ARN de Coronavirus/química , Microscopía por Crioelectrón , ARN Mensajero/química , ARN Viral/química , SARS-CoV-2/química , Proteinas del Complejo de Replicasa Viral/química , Secuencia de Aminoácidos , Coronavirus/química , Coronavirus/clasificación , Coronavirus/enzimología , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , Metiltransferasas/metabolismo , Modelos Moleculares , ARN Helicasas/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , SARS-CoV-2/enzimología , Alineación de Secuencia , Transcripción Genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
Nucleotide analog inhibitors, including broad-spectrum remdesivir and favipiravir, have shown promise in in vitro assays and some clinical studies for COVID-19 treatment, this despite an incomplete mechanistic understanding of the viral RNA-dependent RNA polymerase nsp12 drug interactions. Here, we examine the molecular basis of SARS-CoV-2 RNA replication by determining the cryo-EM structures of the stalled pre- and post- translocated polymerase complexes. Compared with the apo complex, the structures show notable structural rearrangements happening to nsp12 and its co-factors nsp7 and nsp8 to accommodate the nucleic acid, whereas there are highly conserved residues in nsp12, positioning the template and primer for an in-line attack on the incoming nucleotide. Furthermore, we investigate the inhibition mechanism of the triphosphate metabolite of remdesivir through structural and kinetic analyses. A transition model from the nsp7-nsp8 hexadecameric primase complex to the nsp12-nsp7-nsp8 polymerase complex is also proposed to provide clues for the understanding of the coronavirus transcription and replication machinery.
Asunto(s)
Betacoronavirus/química , Betacoronavirus/enzimología , ARN Polimerasa Dependiente del ARN/química , Proteínas no Estructurales Virales/química , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alanina/farmacología , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Dominio Catalítico , ARN Polimerasa Dependiente de ARN de Coronavirus , Microscopía por Crioelectrón , Modelos Químicos , Modelos Moleculares , ARN Viral/metabolismo , SARS-CoV-2 , Transcripción Genética , Replicación ViralRESUMEN
Despite intensive efforts to discover highly effective treatments to eradicate tuberculosis (TB), it remains as a major threat to global human health. For this reason, new TB drugs directed toward new targets are highly coveted. MmpLs (Mycobacterial membrane proteins Large), which play crucial roles in transporting lipids, polymers and immunomodulators and which also extrude therapeutic drugs, are among the most important therapeutic drug targets to emerge in recent times. Here, crystal structures of mycobacterial MmpL3 alone and in complex with four TB drug candidates, including SQ109 (in Phase 2b-3 clinical trials), are reported. MmpL3 consists of a periplasmic pore domain and a twelve-helix transmembrane domain. Two Asp-Tyr pairs centrally located in this domain appear to be key facilitators of proton-translocation. SQ109, AU1235, ICA38, and rimonabant bind inside the transmembrane region and disrupt these Asp-Tyr pairs. This structural data will greatly advance the development of MmpL3 inhibitors as new TB drugs.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/ultraestructura , Adamantano/análogos & derivados , Adamantano/metabolismo , Antituberculosos/química , Transporte Biológico , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Etilenodiaminas/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/ultraestructura , Compuestos de Fenilurea/metabolismo , Rimonabant/metabolismo , Tuberculosis/microbiologíaRESUMEN
Biological energy currency ATP is produced by F1Fo-ATP synthase. However, the molecular mechanism for human ATP synthase action remains unknown. Here, we present snapshot images for three main rotational states and one substate of human ATP synthase using cryoelectron microscopy. These structures reveal that the release of ADP occurs when the ß subunit of F1Fo-ATP synthase is in the open conformation, showing how ADP binding is coordinated during synthesis. The accommodation of the symmetry mismatch between F1 and Fo motors is resolved by the torsional flexing of the entire complex, especially the γ subunit, and the rotational substep of the c subunit. Water molecules are identified in the inlet and outlet half-channels, suggesting that the proton transfer in these two half-channels proceed via a Grotthus mechanism. Clinically relevant mutations are mapped to the structure, showing that they are mainly located at the subunit-subunit interfaces, thus causing instability of the complex.
Asunto(s)
Adenosina Trifosfato , ATPasas de Translocación de Protón , Humanos , Microscopía por Crioelectrón , Adenosina Trifosfato/metabolismo , ATPasas de Translocación de Protón/química , Conformación ProteicaRESUMEN
Bedaquiline (BDQ), a first-in-class diarylquinoline anti-tuberculosis drug, and its analogue, TBAJ-587, prevent the growth and proliferation of Mycobacterium tuberculosis by inhibiting ATP synthase1,2. However, BDQ also inhibits human ATP synthase3. At present, how these compounds interact with either M. tuberculosis ATP synthase or human ATP synthase is unclear. Here we present cryogenic electron microscopy structures of M. tuberculosis ATP synthase with and without BDQ and TBAJ-587 bound, and human ATP synthase bound to BDQ. The two inhibitors interact with subunit a and the c-ring at the leading site, c-only sites and lagging site in M. tuberculosis ATP synthase, showing that BDQ and TBAJ-587 have similar modes of action. The quinolinyl and dimethylamino units of the compounds make extensive contacts with the protein. The structure of human ATP synthase in complex with BDQ reveals that the BDQ-binding site is similar to that observed for the leading site in M. tuberculosis ATP synthase, and that the quinolinyl unit also interacts extensively with the human enzyme. This study will improve researchers' understanding of the similarities and differences between human ATP synthase and M. tuberculosis ATP synthase in terms of the mode of BDQ binding, and will allow the rational design of novel diarylquinolines as anti-tuberculosis drugs.
Asunto(s)
Antituberculosos , Diarilquinolinas , Imidazoles , ATPasas de Translocación de Protón Mitocondriales , Mycobacterium tuberculosis , Piperidinas , Piridinas , Humanos , Antituberculosos/farmacología , Antituberculosos/química , Sitios de Unión , Microscopía por Crioelectrón , Diarilquinolinas/química , Diarilquinolinas/farmacología , Imidazoles/química , Imidazoles/farmacología , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/ultraestructura , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/efectos de los fármacos , Piperidinas/química , Piperidinas/farmacología , Subunidades de Proteína/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/antagonistas & inhibidores , Piridinas/química , Piridinas/farmacologíaRESUMEN
Nirmatrelvir is a specific antiviral drug that targets the main protease (Mpro) of SARS-CoV-2 and has been approved to treat COVID-191,2. As an RNA virus characterized by high mutation rates, whether SARS-CoV-2 will develop resistance to nirmatrelvir is a question of concern. Our previous studies have shown that several mutational pathways confer resistance to nirmatrelvir, but some result in a loss of viral replicative fitness, which is then compensated for by additional alterations3. The molecular mechanisms for this observed resistance are unknown. Here we combined biochemical and structural methods to demonstrate that alterations at the substrate-binding pocket of Mpro can allow SARS-CoV-2 to develop resistance to nirmatrelvir in two distinct ways. Comprehensive studies of the structures of 14 Mpro mutants in complex with drugs or substrate revealed that alterations at the S1 and S4 subsites substantially decreased the level of inhibitor binding, whereas alterations at the S2 and S4' subsites unexpectedly increased protease activity. Both mechanisms contributed to nirmatrelvir resistance, with the latter compensating for the loss in enzymatic activity of the former, which in turn accounted for the restoration of viral replicative fitness, as observed previously3. Such a profile was also observed for ensitrelvir, another clinically relevant Mpro inhibitor. These results shed light on the mechanisms by which SARS-CoV-2 evolves to develop resistance to the current generation of protease inhibitors and provide the basis for the design of next-generation Mpro inhibitors.
Asunto(s)
Antivirales , Farmacorresistencia Viral , SARS-CoV-2 , Humanos , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , COVID-19/virología , Lactamas , Leucina , Nitrilos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , SARS-CoV-2/genética , SARS-CoV-2/crecimiento & desarrollo , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Mutación , Especificidad por Sustrato , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/genética , Proteasas 3C de Coronavirus/metabolismo , Replicación Viral/efectos de los fármacos , Diseño de Fármacos , ProlinaRESUMEN
The development of vaccines, which induce effective immune responses while ensuring safety and affordability, remains a substantial challenge. In this study, we proposed a vaccine model of a restructured "head-to-tail" dimer to efficiently stimulate B cell response. We also demonstrate the feasibility of using this model to develop a paramyxovirus vaccine through a low-cost rice endosperm expression system. Crystal structure and small-angle X-ray scattering data showed that the restructured hemagglutinin-neuraminidase (HN) formed tetramers with fully exposed quadruple receptor binding domains and neutralizing epitopes. In comparison with the original HN antigen and three traditional commercial whole virus vaccines, the restructured HN facilitated critical epitope exposure and initiated a faster and more potent immune response. Two-dose immunization with 0.5 µg of the restructured antigen (equivalent to one-127th of a rice grain) and one-dose with 5 µg completely protected chickens against a lethal challenge of the virus. These results demonstrate that the restructured HN from transgenic rice seeds is safe, effective, low-dose useful, and inexpensive. We provide a plant platform and a simple restructured model for highly effective vaccine development.
Asunto(s)
Oryza , Paramyxovirinae , Vacunas Virales , Animales , Pollos , Virus de la Enfermedad de Newcastle , Oryza/genética , Diseño Universal , Epítopos , Anticuerpos AntiviralesRESUMEN
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.
Asunto(s)
Acetolactato Sintasa/química , Arabidopsis/enzimología , Saccharomyces cerevisiae/enzimología , Acetolactato Sintasa/metabolismo , Adenosina Trifosfato/metabolismo , Aminoácidos de Cadena Ramificada/biosíntesis , Dominio Catalítico , Activación Enzimática , Evolución Molecular , Retroalimentación Fisiológica , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Mycobacterium tuberculosis/enzimología , Unión Proteica , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Valina/metabolismoRESUMEN
Mycobacterium tuberculosis is an intracellular pathogen that uses several strategies to interfere with the signalling functions of host immune molecules. Many other bacterial pathogens exploit the host ubiquitination system to promote pathogenesis1,2, but whether this same system modulates the ubiquitination of M. tuberculosis proteins is unknown. Here we report that the host E3 ubiquitin ligase ANAPC2-a core subunit of the anaphase-promoting complex/cyclosome-interacts with the mycobacterial protein Rv0222 and promotes the attachment of lysine-11-linked ubiquitin chains to lysine 76 of Rv0222 in order to suppress the expression of proinflammatory cytokines. Inhibition of ANAPC2 by specific short hairpin RNA abolishes the inhibitory effect of Rv0222 on proinflammatory responses. Moreover, mutation of the ubiquitination site on Rv0222 impairs the inhibition of proinflammatory cytokines by Rv0222 and reduces virulence during infection in mice. Mechanistically, lysine-11-linked ubiquitination of Rv0222 by ANAPC2 facilitates the recruitment of the protein tyrosine phosphatase SHP1 to the adaptor protein TRAF6, preventing the lysine-63-linked ubiquitination and activation of TRAF6. Our findings identify a previously unrecognized mechanism that M. tuberculosis uses to suppress host immunity, and provide insights relevant to the development of effective immunomodulators that target M. tuberculosis.
Asunto(s)
Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Ubiquitinación , Ciclosoma-Complejo Promotor de la Anafase/química , Animales , Subunidad Apc2 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Células Cultivadas , Citocinas/antagonistas & inhibidores , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Lisina/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/antagonistas & inhibidores , Factor 6 Asociado a Receptor de TNF/metabolismo , Factor de Transcripción AP-1/metabolismo , Tuberculosis/microbiología , Virulencia/inmunologíaRESUMEN
A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the aetiological agent responsible for the 2019-2020 viral pneumonia outbreak of coronavirus disease 2019 (COVID-19)1-4. Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here we describe the results of a programme that aimed to rapidly discover lead compounds for clinical use, by combining structure-assisted drug design, virtual drug screening and high-throughput screening. This programme focused on identifying drug leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-25,6. We identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then determined the crystal structure of Mpro of SARS-CoV-2 in complex with this compound. Through a combination of structure-based virtual and high-throughput screening, we assayed more than 10,000 compounds-including approved drugs, drug candidates in clinical trials and other pharmacologically active compounds-as inhibitors of Mpro. Six of these compounds inhibited Mpro, showing half-maximal inhibitory concentration values that ranged from 0.67 to 21.4 µM. One of these compounds (ebselen) also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.
Asunto(s)
Betacoronavirus/química , Cisteína Endopeptidasas/química , Descubrimiento de Drogas/métodos , Modelos Moleculares , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Antivirales/química , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , COVID-19 , Células Cultivadas/virología , Proteasas 3C de Coronavirus , Infecciones por Coronavirus/enzimología , Infecciones por Coronavirus/virología , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Humanos , Pandemias , Neumonía Viral/enzimología , Neumonía Viral/virología , Inhibidores de Proteasas/farmacología , Estructura Terciaria de Proteína , SARS-CoV-2RESUMEN
Trehalose plays a crucial role in the survival and virulence of the deadly human pathogen Mycobacterium tuberculosis (Mtb). The type I ATP-binding cassette (ABC) transporter LpqY-SugABC is the sole pathway for trehalose to enter Mtb. The substrate-binding protein, LpqY, which forms a stable complex with the translocator SugABC, recognizes and captures trehalose and its analogues in the periplasmic space, but the precise molecular mechanism for this process is still not well understood. This study reports a 3.02-Å cryoelectron microscopy structure of trehalose-bound Mtb LpqY-SugABC in the pretranslocation state, a crystal structure of Mtb LpqY in a closed form with trehalose bound and five crystal structures of Mtb LpqY in complex with different trehalose analogues. These structures, accompanied by substrate-stimulated ATPase activity data, reveal how LpqY recognizes and binds trehalose and its analogues, and highlight the flexibility in the substrate binding pocket of LpqY. These data provide critical insights into the design of trehalose analogues that could serve as potential molecular probe tools or as anti-TB drugs.
Asunto(s)
Mycobacterium tuberculosis , Humanos , Microscopía por Crioelectrón , Trehalosa , Transportadoras de Casetes de Unión a ATP , Sondas MolecularesRESUMEN
Arabinogalactan (AG) is an essential cell wall component in mycobacterial species, including the deadly human pathogen Mycobacterium tuberculosis. It plays a pivotal role in forming the rigid mycolyl-AG-peptidoglycan core for in vitro growth. AftA is a membrane-bound arabinosyltransferase and a key enzyme involved in AG biosynthesis which bridges the assembly of the arabinan chain to the galactan chain. It is known that AftA catalyzes the transfer of the first arabinofuranosyl residue from the donor decaprenyl-monophosphoryl-arabinose to the mature galactan chain (i.e., priming); however, the priming mechanism remains elusive. Herein, we report the cryo-EM structure of Mtb AftA. The detergent-embedded AftA assembles as a dimer with an interface maintained by both the transmembrane domain (TMD) and the soluble C-terminal domain (CTD) in the periplasm. The structure shows a conserved glycosyltransferase-C fold and two cavities converging at the active site. A metal ion participates in the interaction of TMD and CTD of each AftA molecule. Structural analyses combined with functional mutagenesis suggests a priming mechanism catalyzed by AftA in Mtb AG biosynthesis. Our data further provide a unique perspective into anti-TB drug discovery.
Asunto(s)
Mycobacterium tuberculosis , Humanos , Galactanos , Pentosiltransferasa/genéticaRESUMEN
Human complex II is a key protein complex that links two essential energy-producing processes: the tricarboxylic acid cycle and oxidative phosphorylation. Deficiencies due to mutagenesis have been shown to cause mitochondrial disease and some types of cancers. However, the structure of this complex is yet to be resolved, hindering a comprehensive understanding of the functional aspects of this molecular machine. Here, we have determined the structure of human complex II in the presence of ubiquinone at 2.86 Å resolution by cryoelectron microscopy, showing it comprises two water-soluble subunits, SDHA and SDHB, and two membrane-spanning subunits, SDHC and SDHD. This structure allows us to propose a route for electron transfer. In addition, clinically relevant mutations are mapped onto the structure. This mapping provides a molecular understanding to explain why these variants have the potential to produce disease.
Asunto(s)
Estructura Cuaternaria de Proteína , Humanos , Modelos Moleculares , Mutación , Microscopía por CrioelectrónRESUMEN
The enzyme protochlorophyllide oxidoreductase (POR) catalyses a light-dependent step in chlorophyll biosynthesis that is essential to photosynthesis and, ultimately, all life on Earth1-3. POR, which is one of three known light-dependent enzymes4,5, catalyses reduction of the photosensitizer and substrate protochlorophyllide to form the pigment chlorophyllide. Despite its biological importance, the structural basis for POR photocatalysis has remained unknown. Here we report crystal structures of cyanobacterial PORs from Thermosynechococcus elongatus and Synechocystis sp. in their free forms, and in complex with the nicotinamide coenzyme. Our structural models and simulations of the ternary protochlorophyllide-NADPH-POR complex identify multiple interactions in the POR active site that are important for protochlorophyllide binding, photosensitization and photochemical conversion to chlorophyllide. We demonstrate the importance of active-site architecture and protochlorophyllide structure in driving POR photochemistry in experiments using POR variants and protochlorophyllide analogues. These studies reveal how the POR active site facilitates light-driven reduction of protochlorophyllide by localized hydride transfer from NADPH and long-range proton transfer along structurally defined proton-transfer pathways.
Asunto(s)
Clorofila/biosíntesis , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Synechococcus/enzimología , Synechocystis/enzimología , Catálisis , Clorofila/química , Estructura Molecular , Fotoquímica , Protoclorofilida/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key enzyme, which extensively digests CoV replicase polyproteins essential for viral replication and transcription, making it an attractive target for antiviral drug development. However, the molecular mechanism of how Mpro of SARS-CoV-2 digests replicase polyproteins, releasing the nonstructural proteins (nsps), and its substrate specificity remain largely unknown. Here, we determine the high-resolution structures of SARS-CoV-2 Mpro in its resting state, precleavage state, and postcleavage state, constituting a full cycle of substrate cleavage. The structures show the delicate conformational changes that occur during polyprotein processing. Further, we solve the structures of the SARS-CoV-2 Mpro mutant (H41A) in complex with six native cleavage substrates from replicase polyproteins, and demonstrate that SARS-CoV-2 Mpro can recognize sequences as long as 10 residues but only have special selectivity for four subsites. These structural data provide a basis to develop potent new inhibitors against SARS-CoV-2.
Asunto(s)
Proteasas 3C de Coronavirus , ARN Polimerasa Dependiente de ARN de Coronavirus , SARS-CoV-2 , Antivirales/química , Proteasas 3C de Coronavirus/química , ARN Polimerasa Dependiente de ARN de Coronavirus/química , ARN Polimerasa Dependiente de ARN de Coronavirus/genética , Poliproteínas/química , Conformación Proteica , Proteolisis , SARS-CoV-2/enzimología , Especificidad por Sustrato/genéticaRESUMEN
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.