RESUMO
SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral Mpro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. The lack of structural information for intermediary forms of Mpro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 Mpro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the Mpro bound to its endogenous N and C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.
Assuntos
Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , SARS-CoV-2/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Domínio Catalítico/fisiologia , Cristalografia por Raios X/métodos , Dimerização , HumanosRESUMO
WONKA is a tool for the systematic analysis of an ensemble of protein-ligand structures. It makes the identification of conserved and unusual features within such an ensemble straightforward. WONKA uses an intuitive workflow to process structural co-ordinates. Ligand and protein features are summarised and then presented within an interactive web application. WONKA's power in consolidating and summarising large amounts of data is described through the analysis of three bromodomain datasets. Furthermore, and in contrast to many current methods, WONKA relates analysis to individual ligands, from which we find unusual and erroneous binding modes. Finally the use of WONKA as an annotation tool to share observations about structures is demonstrated. WONKA is freely available to download and install locally or can be used online at http://wonka.sgc.ox.ac.uk.
Assuntos
Desenho de Fármacos , Proteínas/química , Software , Bases de Dados de Proteínas , Histona Acetiltransferases , Chaperonas de Histonas , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas/metabolismo , Fatores Genéricos de Transcrição , Fluxo de TrabalhoRESUMO
Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate on the pantothenate (vitamin B(5)) biosynthetic pathway. The Escherichia coli panE gene encoding KPR was cloned and expressed at high levels as the native and selenomethionine-substituted (SeMet) proteins. Both native and SeMet recombinant proteins were purified by three chromatographic steps, to yield pure proteins. The wild-type enzyme was found to have a K(M)(NADPH) of 20 microM, a K(M)(ketopantoate) of 60 microM, and a k(cat) of 40 s(-1). Regular prismatic KPR crystals were prepared using the hanging drop technique. They belonged to the tetragonal space group P4(2)2(1)2, with cell parameters: a = b = 103.7 A and c = 55.7 A, accommodating one enzyme molecule per asymmetric unit. The structure of KPR was determined by the multiwavelength anomalous dispersion method using the SeMet protein, for which data were collected to 2.3 A resolution. The native data were collected to 1.7 A resolution and used to refine the final structure. The secondary structure comprises 12 alpha-helices, three 3(10)-helices, and 11 beta-strands. The enzyme is monomeric and has two domains separated by a cleft. The N-terminal domain has an alphabeta-fold of the Rossmann type. The C-terminal domain (residues 170-291) is composed of eight alpha-helices. KPR is shown to be a member of the 6-phosphogluconate dehydrogenase C-terminal domain-like superfamily. A model for the ternary enzyme-NADPH-ketopantoate ternary complex provides a rationale for kinetic data reported for specific site-directed mutants.
Assuntos
Oxirredutases do Álcool/química , Escherichia coli/enzimologia , Selenometionina/química , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Primers do DNA/química , Escherichia coli/genética , Expressão Gênica , Cinética , Dados de Sequência Molecular , NADP/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: Pantothenate synthetase (EC 6.3.2.1) is the last enzyme of the pathway of pantothenate (vitamin B(5)) synthesis. It catalyzes the condensation of pantoate with beta-alanine in an ATP-dependent reaction. RESULTS: We describe the overexpression, purification, and crystal structure of recombinant pantothenate synthetase from E. coli. The structure was solved by a selenomethionine multiwavelength anomalous dispersion experiment and refined against native data to a final R(cryst) of 22.6% (R(free) = 24.9%) at 1.7 A resolution. The enzyme is dimeric, with two well-defined domains per protomer: the N-terminal domain, a Rossmann fold, contains the active site cavity, with the C-terminal domain forming a hinged lid. CONCLUSIONS: The N-terminal domain is structurally very similar to class I aminoacyl-tRNA synthetases and is thus a member of the cytidylyltransferase superfamily. This relationship has been used to suggest the location of the ATP and pantoate binding sites and the nature of hinge bending that leads to the ternary enzyme-pantoate-ATP complex.
Assuntos
Escherichia coli/enzimologia , Peptídeo Sintases/química , Trifosfato de Adenosina/metabolismo , Cristalografia por Raios X , Dimerização , Expressão Gênica , Peptídeo Sintases/classificação , Peptídeo Sintases/genética , Peptídeo Sintases/isolamento & purificação , Estrutura Secundária de Proteína , Soluções , Especificidade por SubstratoRESUMO
The decarboxylation of L-aspartate by E. coli L-aspartate-alpha-decarboxylase (ADC) is shown to occur with retention of configuration; analysis of the protein structure identifies Tyr58 as the proton donor in the decarboxylation mechanism.
Assuntos
Glutamato Descarboxilase/química , Glutamato Descarboxilase/metabolismo , Tirosina/metabolismo , Sítios de Ligação , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Prótons , EstereoisomerismoRESUMO
Fibroblast growth factors (FGFs) are a large family of structurally related proteins with a wide range of physiological and pathological activities. Signal transduction requires association of FGF with its receptor tyrosine kinase (FGFR) and heparan sulphate proteoglycan in a specific complex on the cell surface. Direct involvement of the heparan sulphate glycosaminoglycan polysaccharide in the molecular association between FGF and its receptor is essential for biological activity. Although crystal structures of binary complexes of FGF-heparin and FGF-FGFR have been described, the molecular architecture of the FGF signalling complex has not been elucidated. Here we report the crystal structure of the FGFR2 ectodomain in a dimeric form that is induced by simultaneous binding to FGF1 and a heparin decasaccharide. The complex is assembled around a central heparin molecule linking two FGF1 ligands into a dimer that bridges between two receptor chains. The asymmetric heparin binding involves contacts with both FGF1 molecules but only one receptor chain. The structure of the FGF1-FGFR2-heparin ternary complex provides a structural basis for the essential role of heparan sulphate in FGF signalling.
Assuntos
Fator 2 de Crescimento de Fibroblastos/química , Heparina/química , Receptores Proteína Tirosina Quinases/química , Receptores de Fatores de Crescimento de Fibroblastos/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli , Fator 1 de Crescimento de Fibroblastos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparina/metabolismo , Humanos , Ligantes , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes/químicaRESUMO
The structure of L-aspartate-alpha-decarboxylase from E. coli has been determined at 2.2 A resolution. The enzyme is a tetramer with pseudofour-fold rotational symmetry. The subunits are six-stranded beta-barrels capped by small alpha-helices at each end. The active sites are located between adjacent subunits. The electron density provides evidence for catalytic pyruvoyl groups at three active sites and an ester at the fourth. The ester is an intermediate in the autocatalytic self-processing leading to formation of the pyruvoyl group. This unprecedented structure provides novel insights into the general phenomenon of protein processing.