RESUMEN
Hepatitis B virus (HBV) X protein, HBx, interacts with anti-apoptotic Bcl-2 and Bcl-xL proteins through its BH3-like motif to promote HBV replication and cytotoxicity. Here we report the crystal structure of HBx BH3-like motif in complex with Bcl-xL where the BH3-like motif adopts a short α-helix to snuggle into a hydrophobic pocket in Bcl-xL via its noncanonical Trp120 residue and conserved Leu123 residue. This binding pocket is ~2 Å away from the canonical BH3-only binding pocket in structures of Bcl-xL with proapoptotic BH3-only proteins. Mutations altering Trp120 and Leu123 in HBx impair its binding to Bcl-xL in vitro and HBV replication in vivo, confirming the importance of this motif to HBV. A HBx BH3-like peptide, HBx-aa113-135, restores HBV replication from a HBx-null HBV replicon, while a shorter peptide, HBx-aa118-127, inhibits HBV replication. These results provide crucial structural and functional insights into drug designs for inhibiting HBV replication and treating HBV patients.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/química , Virus de la Hepatitis B/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-bcl-2/química , Transactivadores/química , Transactivadores/fisiología , Proteína bcl-X/química , Animales , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Células Hep G2 , Virus de la Hepatitis B/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Mutación , Unión Proteica , Transactivadores/genética , Proteínas Reguladoras y Accesorias Virales , Replicación Viral/fisiologíaRESUMEN
The ability to obtain a large number of human pluripotent stem cells (hPSCs) under chemically defined conditions plays a key role in clinical application of hPSCs. Chemically defined, economical and effective synthetic peptide displaying surfaces should be the optimal choice for clinical applications involving hPSCs. However, synthetic peptide displaying surfaces are worse than Matrigel surface in supporting cell adhesion and self-renewal. Moreover, the correlations between peptide amino acid sequences and the ability of peptides to support cell survival has never been investigated in hPSCs. In this study, we focused on the Arg-Gly-Asp (RGD) sequence and integrin receptors, which constitute the major recognition system for cell adhesion. Several new RGD-containing peptides were designed by altering the amino acids surrounding the RGD sequence. We investigated the ability of these peptides to sustain hPSC survival, and identified the Ac-KGGPQVTRGDTYRAY sequence, which was capable of supporting cell reprogramming, long-term self-renewal and lineage differentiation. In addition, this report demonstrates that the introduction of mutations in the amino acids surrounding the RGD sequence is a good strategy to design peptides that display excellent adhesion properties and promote hPSC self-renewal. Our results will help improve the current understanding of the mechanisms by which RGD-containing peptides exhibit different abilities in sustaining hPSC culture, and will promote clinical application of both peptide displaying surfaces and hPSCs.
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Oligopéptidos/farmacología , Células Madre Pluripotentes/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Oligopéptidos/química , Tamaño de la Partícula , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
In humans, primary microRNA (pri-miRNA) processing starts from precise cleavage of the stem loop, which is catalyzed by the Drosha-DGCR8 complex. However, the significant inconsistencies in the expression levels among primary, precursor, and mature miRNAs clearly indicate that many other factors may be involved in this regulation. Here, we utilize a newly developed RNA affinity technique to isolate such factors. In this study, a tRNA-scaffolded aptamer (tRSA)-based RNA affinity tag, by directly fusing primary let-7 miRNA to the 3'-end of tRSA, is employed to pull down the protein components specifically binding to pri-let-7. We show that La protein binds to pri-let-7 via its La motif and significantly promotes the processing efficiency of pri-let-7 in vitro and in cells. In addition, we demonstrate that La protein is associated with DGCR8, but not Drosha, in an RNA-dependent manner. Interestingly, the RNA binding capacity of La motif is important for miRNA processing. Hence, we propose that La protein is an important microprocessor component regulating miRNA processing efficiency by association with DGCR8 to regulate formation of the DGCR8-Drosha complex for miRNA processing.
Asunto(s)
MicroARNs/metabolismo , Fosfoproteínas/metabolismo , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Células HEK293 , Humanos , MicroARNs/química , Modelos Moleculares , Conformación de Ácido Nucleico , Fosfoproteínas/genética , Interferencia de ARN , Precursores del ARN/química , Procesamiento Postranscripcional del ARN , Regulación hacia ArribaRESUMEN
We have previously shown that a non-structural protein 1 (NS1)-binding monoclonal antibody, termed as 2H6, can significantly reduce influenza A virus (IAV) replication when expressed intracellularly. In this study, we further showed that 2H6 binds stronger to the NS1 of H5N1 than A/Puerto Rico/8/1934(H1N1) because of an amino acid difference at residue 48. A crystal structure of 2H6 fragment antigen-binding (Fab) has also been solved and docked onto the NS1 structure to reveal the contacts between specific residues at the interface of antibody-antigen complex. In one of the models, the predicted molecular contacts between residues in NS1 and 2H6-Fab correlate well with biochemical results. Taken together, residues N48 and T49 in H5N1 NS1 act cooperatively to maintain a strong interaction with mAb 2H6 by forming hydrogen bonds with residues found in the heavy chain of the antibody. Interestingly, the pandemic H1N1-2009 and the majority of seasonal H3N2 circulating in humans since 1968 has N48 in NS1, suggesting that mAb 2H6 could bind to most of the currently circulating seasonal influenza A virus strains. Consistent with the involvement of residue T49, which is well-conserved, in RNA binding, mAb 2H6 was also found to inhibit the interaction between NS1 and double-stranded RNA.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Virus de la Influenza A/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Células A549 , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Antígenos Virales/metabolismo , Sitios de Unión , Aves/virología , Cristalografía por Rayos X , Perros , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Subtipo H5N1 del Virus de la Influenza A/metabolismo , Células de Riñón Canino Madin Darby , Mutación/genética , Unión Proteica , ARN Bicatenario/metabolismo , Replicación ViralRESUMEN
BACKGROUND: Foot-and-mouth disease (FMD) is an acute, highly contagious disease that infects cloven-hoofed animals. Vaccination is an effective means of preventing and controlling FMD. Compared to conventional inactivated FMDV vaccines, the format of FMDV virus-like particles (VLPs) as a non-replicating particulate vaccine candidate is a promising alternative. RESULTS: In this study, we have developed a co-expression system in E. coli, which drove the expression of FMDV capsid proteins (VP0, VP1, and VP3) in tandem by a single plasmid. The co-expressed FMDV capsid proteins (VP0, VP1, and VP3) were produced in large scale by fermentation at 10 L scale and the chromatographic purified capsid proteins were auto-assembled as VLPs in vitro. Cattle vaccinated with a single dose of the subunit vaccine, comprising in vitro assembled FMDV VLP and adjuvant, developed FMDV-specific antibody response (ELISA antibodies and neutralizing antibodies) with the persistent period of 6 months. Moreover, cattle vaccinated with the subunit vaccine showed the high protection potency with the 50 % bovine protective dose (PD50) reaching 11.75 PD50 per dose. CONCLUSIONS: Our data strongly suggest that in vitro assembled recombinant FMDV VLPs produced from E. coli could function as a potent FMDV vaccine candidate against FMDV Asia1 infection. Furthermore, the robust protein expression and purification approaches described here could lead to the development of industrial level large-scale production of E. coli-based VLPs against FMDV infections with different serotypes.
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Enfermedades de los Bovinos/prevención & control , Enfermedades de los Bovinos/virología , Fiebre Aftosa/prevención & control , Fiebre Aftosa/virología , Vacunas de Partículas Similares a Virus/biosíntesis , Animales , Técnicas de Cultivo Celular por Lotes/métodos , Bovinos , Escherichia coli/genética , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vacunas de Partículas Similares a Virus/genética , Vacunas de Partículas Similares a Virus/uso terapéuticoRESUMEN
We report the strategies leading to the large-production of soluble non-tag full-length porcine circovirus type 2 (PCV2) Cap protein in Escherichia coli. Under neutral pH condition, the purified recombinant Cap protein derived from E. coli expression self-assembles into homogenous round virus-like particle at the similar size of that of the intact PCV2 virus, which is further characterized by Cryo-EM single particle structure determined at 4.5Å. The engineered PCV2 rCap VLP was tested as a subunit vaccine for the protective efficacy against PCV2 challenge on 3-week old piglets. Similar to commercial available PCV2 vaccine, the Cap VLP-immunized piglets developed specific antibody-mediated response and were protected from the virulent SH PCV2 strain challenge. Hence, the production of E. coli based PCV2Cap-VLP could be applied as a cost-friendly and effective subunit vaccine to control PCV2 spreading in developing countries.
Asunto(s)
Infecciones por Circoviridae/prevención & control , Infecciones por Circoviridae/veterinaria , Circovirus/inmunología , Escherichia coli/genética , Vacunas de Partículas Similares a Virus/administración & dosificación , Animales , Anticuerpos Antivirales/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Infecciones por Circoviridae/inmunología , Escherichia coli/metabolismo , Expresión Génica , Porcinos , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/metabolismo , Vacunas de Partículas Similares a Virus/genética , Vacunas de Partículas Similares a Virus/metabolismo , Ensamble de VirusRESUMEN
OBJECTIVE: This study aimed to investigate the therapeutic potential of monoclonal antibody (mAb) against HBV as a novel treatment approach to chronic hepatitis B (CHB) in mouse models. METHODS: Therapeutic effects of mAbs against various epitopes on viral surface protein were evaluated in mice mimicking persistent HBV infection. The immunological mechanisms of mAb-mediated viral clearance were systematically investigated. RESULTS: Among 11 tested mAbs, a novel mAb E6F6 exhibited the most striking therapeutic effects in several HBV-persistent mice. Single-dose administration of E6F6 could profoundly suppress the levels of hepatitis B surface antigen (HBsAg) and HBV DNA for several weeks in HBV-transgenic mice. E6F6 regimen efficiently prevented initial HBV infection, and reduced viral dissemination from infected hepatocytes in human-liver-chimeric mice. E6F6-based immunotherapy facilitated the restoration of anti-HBV T-cell response in hydrodynamic injection (HDI)-based HBV carrier mice. Immunological analyses suggested that the Fcγ receptor-dependent phagocytosis plays a predominant role in E6F6-mediated viral suppression. Molecular analyses suggested that E6F6 recognises an evolutionarily conserved epitope (GPCK(R)TCT) and only forms a smaller antibody-viral particle immune complex with limited interparticle crosslinking when it binds to viral particles. This unique binding characteristic of E6F6 to HBV was possibly associated with its effective in vivo opsonophagocytosis for viral clearance. CONCLUSIONS: These results provided new insight into understanding the therapeutic role and mechanism of antibody against persistent viral infection. The E6F6-like mAbs may provide a novel immunotherapeutic agent against human chronic HBV infection.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos de Superficie de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/tratamiento farmacológico , Inmunoterapia/métodos , Animales , ADN Viral/efectos de los fármacos , Modelos Animales de Enfermedad , Epítopos , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Hepatocitos/virología , Ratones , Ratones Transgénicos , Fagocitosis , Replicación Viral/efectos de los fármacosRESUMEN
In our previous work, a NAD(H)-dependent carbonyl reductase (GoCR) was identified from Gluconobacter oxydans, which showed moderate to high enantiospecificity for the reduction of different kinds of prochiral ketones. In the present study, the crystal structure of GoCR was determined at 1.65Å resolution, and a computational strategy concerning substrate-enzyme docking and all-atom molecular dynamics (MD) simulation was established to help understand the molecular basis of enantiopreference and enantiorecognition for GoCR, and to further guide the design and engineering of GoCR enantioselectivity. For the reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE), three binding pocket residues, Cys93, Tyr149, and Trp193 were predicted to play a critical role in determining the enantioselectivity. Through site-directed mutagenesis, single-point mutant W193A was constructed and proved to reduce OPBE to ethyl (R)-2-hydroxy-4-phenylbutyrate (R-HPBE) with a significantly improved ee of >99% compared to 43.2% for the wild type (WT). Furthermore, double mutant C93V/Y149A was proved to even invert the enantioselectivity of GoCR to afford S-HPBE at 79.8% ee.
Asunto(s)
Aldehído Reductasa/química , Gluconobacter oxydans/enzimología , Ingeniería de Proteínas/métodos , Aldehído Reductasa/genética , Aldehído Reductasa/aislamiento & purificación , Aldehído Reductasa/metabolismo , Aldo-Ceto Reductasas , Sitios de Unión , Biología Computacional/métodos , Escherichia coli/genética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Fenilbutiratos/metabolismo , Estereoisomerismo , Especificidad por SustratoRESUMEN
In prokaryotes, the CRISPR/Cas system is known to target and degrade invading phages and foreign genetic elements upon subsequent infection. However, the structure and function of many Cas proteins remain largely unknown, due to the high diversity of Cas proteins. Here we report 3 crystal structures of Archaeoglobus fulgidus Csx3 (AfCsx3) in free form, in complex with manganese ions and in complex with a single-stranded RNA (ssRNA) fragment, respectively. AfCsx3 harbors a ferredoxin-like fold and forms dimer both in the crystal and in solution. Our structure-based biochemical analysis demonstrates that the RNA binding sites and cleavage sites are located at 2 separate surfaces within the AfCsx3 dimer, suggesting a model to bind, tether and cleave the incoming RNA substrate. In addition, AfCsx3 displays robust 3'-deadenylase activity in the presence of manganese ions, which strongly suggests that AfCsx3 functions as a deadenylation exonuclease. Taken together, our results indicate that AfCsx3 is a Cas protein involved in RNA deadenylation and provide a framework for understanding the role of AfCsx3 in the Type III-B CRISPR/Cas system.
Asunto(s)
Proteínas Asociadas a CRISPR/química , Exorribonucleasas/química , Manganeso/química , Conformación Molecular , Ribonucleasas/química , Archaeoglobus fulgidus , Proteínas Asociadas a CRISPR/metabolismo , ARN de Archaea/químicaRESUMEN
Coenzyme engineering, especially for altered coenzyme specificity, has been a research hotspot for more than a decade. In the present study, a novel computational strategy that enhances the hydrogen-bond interaction between an enzyme and a coenzyme was developed and utilized to alter the coenzyme preference. This novel computational strategy only required the structure of the target enzyme. No other homologous enzymes were needed to achieve alteration in the coenzyme preference of a certain enzyme. Using our novel strategy, Gox2181 was reconstructed from exhibiting complete NADPH preference to exhibiting dual cofactor specificity for NADH and NADPH. Structure-guided Gox2181 mutants were designed in silico and molecular dynamics simulations were performed to evaluate the strength of hydrogen-bond interactions between the enzyme and the coenzyme NADPH. Three Gox2181 mutants displaying high structure stability and structural compatibility to NADH/NADPH were chosen for experimental confirmation. Among the three Gox2181 mutants, Gox2181-Q20R&D43S showed the highest enzymatic activity by utilizing NADPH as its coenzyme, which was even better than the wild-type enzyme. In addition, isothermal titration calorimetry analysis further verified that Gox2181-Q20R&D43S was able to interact with NADPH but the wild-type enzyme could not. This novel computational strategy represents an insightful approach for altering the cofactor preference of target enzymes.
Asunto(s)
Coenzimas/metabolismo , Modelos Moleculares , Proteínas Mutantes/metabolismo , NADP/metabolismo , NAD/metabolismo , Oxidorreductasas/metabolismo , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Coenzimas/química , Biología Computacional , Secuencia Conservada , Bases de Datos de Proteínas , Estabilidad de Enzimas , Sistemas Especialistas , Gluconobacter oxydans/enzimología , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , NAD/química , NADP/química , Oxidorreductasas/química , Oxidorreductasas/genética , Conformación ProteicaRESUMEN
CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)-mediated defense against invading nucleic acids is a process recently discovered in prokaryotes, which includes recognition and incorporation of invading genetic elements, transcription and processing of CRISPR-RNA (crRNA) and targeting the invaders through base pair recognition. In the type I-E CRISPR-Cas system, Cse2 is proposed to provide a platform to facilitate the targeting of the invading dsDNA by crRNA. Here we report the crystal structure of Meiothermus ruber Cse2 at 2.8Å. M. ruber Cse2 adopts an α-helical bundle scaffold, harbors a positive surface for nucleic acid binding and a conserved dimer interface with strikingly low buried surface area. M. ruber Cse2 selectively binds to G-rich crRNA sequence, which is stripped off from the Cse2-crRNA and Cascade-crRNA complexes by ssDNA or dsDNA with complementary sequence. Stable M. ruber Cascade is readily formed by co-expression of M. ruber Cascade proteins together with G-rich crRNA in vitro. Docking of M. ruber Cse2 structures into the Escherichia coli Cascade Cryo-EM envelope reveals a curved elongated shallow groove for ssRNA binding, which adopts a similar dimer interface discovered by high-resolution crystal structure of Cse2 within E. Coli Cascade. Taken together, our data provides the structural insights into crRNA G-rich sequence recognition by M. ruber Cse2 and reveals the potential structural mechanism for M. ruber Cascade assembly and function.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sistemas CRISPR-Cas/genética , Bacterias Grampositivas/genética , Bacterias Grampositivas/metabolismo , Modelos Moleculares , ARN/metabolismo , Proteínas Bacterianas/genética , Northern Blotting , Cartilla de ADN/genética , Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética , Conformación Proteica , UltracentrifugaciónRESUMEN
The emergence of resistant influenza A viruses highlights the continuous requirement of new antiviral drugs that can treat the viral infection. Non-structural 1 (NS1) protein, an indispensable component for efficient virus replication, can be used as a potential target for generating new antiviral agents. Here, we study the interaction of 2H6 monoclonal antibody with NS1 protein and also determine whether influenza virus replication can be inhibited by blocking NS1. The 2H6-antigen binding fragment (Fab) forms a multimeric complex with the NS1 RNA-binding domain (RBD). T49, a residue which forms a direct hydrogen bond with double stranded RNA, in NS1 protein was found to be critical for its interaction with 2H6 antibody. NS1(RBD) has high affinity to 2H6 with KD of 43.5±4.24nM whereas NS1(RBD)-T49A has more than 250 times lower affinity towards 2H6. Interestingly, the intracellular expression of 2H6-single-chain variable fragment (scFv) in mammalian cells caused a reduction in viral growth and the M1 viral protein level was significantly reduced in 2H6-scFv transfected cells in comparison to vector transfected cells at 12h post infection. These results indicate that the tight binding of 2H6 to NS1 could lead to reduction in viral replication and release of progeny virus. In future, 2H6 antibody in combination with other neutralizing antibodies can be used to increase the potency of viral inhibition.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Virus de la Influenza A/inmunología , Virus de la Influenza A/fisiología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/inmunología , Replicación Viral , Sustitución de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Perros , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Treonina/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
We report the crystal structure of old yellow enzyme (OYE) family protein Gox0502 (a.a 1-315) in free form at 3.3 Å. Detailed structural analysis revealed the key residues involved in stereospecific determination of Gox0502, such as Trp66 and Trp100. Structure-based computational analysis suggested the bulky side chains of these tryptophan residues may play important roles in product stereoselectivity. The introduction of Ile or Phe or Tyr mutation significantly reduced the product diastereoselectivity. We hypothesized that less bulky side chains at these critical residues could create additional free space to accommodate intermediates with different conformations. Notably, the introduction of Phe mutation at residue Trp100 increased catalytic activity compared to wild-type Gox0502 toward a set of substrates tested, which suggests that a less bulky Phe side chain at residue W100F may facilitate product release. Therefore, Gox0502 structure could provide useful information to generate desirable OYEs suitable for biotechnological applications in industry.
Asunto(s)
Aldehídos/química , Aldehídos/metabolismo , Gluconobacter oxydans/enzimología , Cetonas/química , Cetonas/metabolismo , NADPH Deshidrogenasa/química , NADPH Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Mononucleótido de Flavina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Alineación de Secuencia , Estereoisomerismo , Especificidad por SustratoRESUMEN
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) defense system is the only adaptive and inheritable immunity found in prokaryotes. The immunity is achieved through a multistep process of adaptation, expression, and interference. In the Type I-E system, interference is mediated by the CRISPR-associated complex for antiviral defense (Cascade), which recognizes invading double-stranded DNA (dsDNA) through the protospacer adjacent motif (PAM) by one of the Cascade components, Cse1. Here, we report the crystal structure of Thermobifida fusca Cse1 at 3.3 Å resolution. T. fusca Cse1 reveals the chair-like two-domain architecture with a well-defined flexible loop, L1, located at the larger N-terminal domain, which was not observed in previous structures of the single Cse1 protein. Structure-based mutagenesis analysis demonstrates that the well-defined flexible loop and a partially conserved structural motif ([FW]-X-[TH]) are involved in PAM binding and recognition, respectively. Moreover, structural docking of T. fusca Cse1 into Escherichia coli Cascade cryoelectron microscopy maps, coupled with structural comparison, reveals a conserved positive patch that is contiguous with Cse2 in the Cascade complex and adjacent to the Cas3 binding site, suggesting its role in R-loop formation/stabilization and the recruitment of Cas3 for target cleavage. Consistent with the structural observation, the introduction of alanine mutations at this positive patch abolished DNA binding activity by Cse1. Taken together, these results suggest that Cse1 is a critical Cascade component involved in Cascade assembly, dsDNA target recognition, R-loop formation, and Cas3 recruitment for target cleavage.
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Actinomycetales/química , Proteínas Fúngicas/química , Actinomycetales/metabolismo , Infecciones por Actinomycetales/microbiología , Secuencia de Aminoácidos , Sitios de Unión , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Cristalografía por Rayos X , ADN de Hongos/metabolismo , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de SecuenciaRESUMEN
Nitrilases are enzymes widely expressed in prokaryotes and eukaryotes that utilize a CysGluLys catalytic triad to hydrolyze non-peptide carbonnitrogen bonds. Nitrilase from Syechocystis sp. Strain PCC6803 (Nit6803) shows hydrolysis activity towards a broad substrate spectrum, ranging from mononitriles to dinitriles and from aromatic nitriles to aliphatic nitriles. Yet, the structural principle of the substrate specificity of this nitrilase is still unknown. We report the crystal structure of Nit6803 at 3.1 Å resolution and propose a structural mechanism of substrate selection. Our mutagenesis data exhibited that the aromaticity of the amino acid at position 146 of Nit6803 is absolutely required for its nitrilase activity towards any substrates tested. Moreover, molecular docking and dynamic simulation analysis indicated that the distance between the sulfhydryl group of the catalytic cysteine residue and the cyano carbon of the substrate plays a crucial role in determining the nitrilase catalytic activity of Nit6803 and its mutants towards different nitrile substrates.
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Aminoácidos/química , Aminoácidos/metabolismo , Aminohidrolasas/química , Aminohidrolasas/metabolismo , Synechocystis/metabolismo , Secuencia de Aminoácidos , Catálisis , Cisteína/química , Cisteína/metabolismo , Hidrólisis , Simulación del Acoplamiento Molecular/métodos , Datos de Secuencia Molecular , Nitrilos/química , Nitrilos/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Rab GTPases, by targeting to specific membrane compartments, play essential roles in membrane trafficking. Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Fsp27 is shown to be required for LD fusion and growth by enriching at LD-LD contact sites. Here, we identify Rab8a as a direct interactor and regulator of Fsp27 in mediating LD fusion in adipocytes. Knockdown of Rab8a in the livers of ob/ob mice results in the accumulation of smaller LDs and lower hepatic lipid levels. Surprisingly, it is the GDP-bound form of Rab8a that exhibits fusion-promoting activity. We further discover AS160 as the GTPase activating protein (GAP) for Rab8a, which forms a ternary complex with Fsp27 and Rab8a to positively regulate LD fusion. MSS4 antagonizes Fsp27-mediated LD fusion activity through Rab8a. Our results have thus revealed a mechanistic signaling circuit controlling LD fusion and fatty liver formation.
Asunto(s)
Adipocitos/metabolismo , Gránulos Citoplasmáticos/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Metabolismo de los Lípidos , Chaperonas Moleculares/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Adipocitos/citología , Animales , Ratones , Ratones Obesos , Chaperonas Moleculares/genética , Células 3T3 NIH , Unión Proteica , Proteínas/metabolismoRESUMEN
Gox2253 from Gluconobacter oxydans belongs to the short-chain dehydrogenases/reductases family, and catalyzes the reduction of heptanal, octanal, nonanal, and decanal with NADPH. To develop a robust working platform to engineer novel G. oxydans oxidoreductases with designed coenzyme preference, we adopted a structure based rational design strategy using computational predictions that considers the number of hydrogen bonds formed between enzyme and docked coenzyme. We report the crystal structure of Gox2253 at 2.6 Å resolution, ternary models of Gox2253 mutants in complex with NADH/short-chain aldehydes, and propose a structural mechanism of substrate selection. Molecular dynamics simulation shows that hydrogen bonds could form between 2'-hydroxyl group in the adenosine moiety of NADH and the side chain of Gox2253 mutant after arginine at position 42 is replaced with tyrosine or lysine. Consistent with the molecular dynamics prediction, Gox2253-R42Y/K mutants can use both NADH and NADPH as a coenzyme. Hence, the strategies here could provide a practical platform to engineer coenzyme selectivity for any given oxidoreductase and could serve as an additional consideration to engineer substrate-binding pockets.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Gluconobacter oxydans/química , Aldehídos/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Dominio Catalítico , Coenzimas/metabolismo , Cristalografía por Rayos X , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutación , NADP/química , NADP/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Conformación Proteica , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
In holometabolous insects, the accumulation and utilization of storage proteins (SPs), including arylphorins and methionine-rich proteins, are critical for the insect metamorphosis. SPs function as amino acids reserves, which are synthesized in fat body, secreted into the larval hemolymph and taken up by fat body shortly before pupation. However, the detailed molecular mechanisms of digestion and utilization of SPs during development are largely unknown. Here, we report the crystal structure of Bombyx mori arylphorins at 2.8 Å, which displays a heterohexameric structural arrangement formed by trimerization of dimers comprising two structural similar arylphorins. Our limited proteolysis assay and microarray data strongly suggest that papain-like proteases are the major players for B. mori arylphorins digestion in vitro and in vivo. Consistent with the biochemical data, dozens of papain cleavage sites are mapped on the surface of the heterohexameric structure of B. mori arylphorins. Hence, our results provide the insightful information to understand the metamorphosis of holometabolous insects at molecular level.
Asunto(s)
Bombyx/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Papaína/química , Papaína/metabolismo , Animales , Cristalografía por Rayos X , Multimerización de ProteínaRESUMEN
Because of the complex mechanisms of enzymatic reactions, no precise and simple method of understanding and controlling the chiral selectivity of enzymes has been developed. However, structure-based rational design is a powerful approach to engineering enzymes with desired catalytic activities. In this work, a simple, structure-based, large-scale in silico design and virtual screening strategy was developed and successfully applied to enzyme engineering. We first performed protein crystallization and X-ray diffraction to determine the structure of lipase LipK107, which is a novel family I.1 lipase displaying activity for both R and S isomers in chiral resolution reactions. The catalytic mechanism of family I.1, which includes LipK107, was ascertained first through comparisons of the sequences and structures of lipases from other families. The binding states of LipK107, including the energy and the conformation of complexes with the R and S enantiomers, have been evaluated by careful biocomputation to figure out the reason for the higher S selectivity. Based on this study, a simple strategy for manipulating the chiral selectivity by modulating a crucial distance in the enzyme-substrate complex and judging virtual mutations in silico is recommended. Then, a novel electrostatic interaction analysis protocol was used to design LipK107 mutants to validate our strategy. Both positive and negative mutations determined using this theoretical protocol have been implemented in wet experiments and were proved to produce the desired enantioselectivity, showing a 176% increase or 50% decrease in enantioselectivity as desired. Because of its accuracy and versatility, the strategy is promising for practical applications.