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1.
Nat Commun ; 11(1): 683, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996686

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

2.
J Phys Chem B ; 123(34): 7365-7371, 2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31365827

RESUMO

The behavior of the structural parameters of DNA considering different levels of methylation in CpG islands is studied by means of full-atom molecular dynamics simulations and electronic circular dichroism, both in an artificial model system and in a gene promoter sequence. It is demonstrated that methylation although intrinsically brings quite local perturbations may, if its level is high enough, induce cooperative effects that strongly modify the DNA backbone torsional parameters altering the helicity as compared to the nonmethylated case. Because methylation of the CpG island is correlated with the regulation of gene expression, understanding the structural modifications induced in DNA is crucial to characterize all the fine equilibria into play in epigenetics phenomena.

3.
Nat Commun ; 10(1): 553, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696828

RESUMO

In the original version of this Article, the final concentration of riboflavin in the supplemented LB medium for recombinant LkcE expression was incorrectly stated as 1 g L-1 (this was the concentration of the stock solution) and should have read 10-50 mg L-1. This error has been corrected in both the PDF and HTML versions of the Article.

4.
Nat Commun ; 9(1): 3998, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30266997

RESUMO

Acquisition of new catalytic activity is a relatively rare evolutionary event. A striking example appears in the pathway to the antibiotic lankacidin, as a monoamine oxidase (MAO) family member, LkcE, catalyzes both an unusual amide oxidation, and a subsequent intramolecular Mannich reaction to form the polyketide macrocycle. We report evidence here for the molecular basis for this dual activity. The reaction sequence involves several essential active site residues and a conformational change likely comprising an interdomain hinge movement. These features, which have not previously been described in the MAO family, both depend on a unique dimerization mode relative to all structurally characterized members. Taken together, these data add weight to the idea that designing new multifunctional enzymes may require changes in both architecture and catalytic machinery. Encouragingly, however, our data also show LkcE to bind alternative substrates, supporting its potential utility as a general cyclization catalyst in synthetic biology.


Assuntos
Proteínas de Bactérias/metabolismo , Macrolídeos/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Streptomyces/metabolismo , Amidas/química , Amidas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Vias Biossintéticas/genética , Macrolídeos/síntese química , Macrolídeos/química , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Conformação Proteica , Multimerização Proteica , Homologia de Sequência de Aminoácidos , Streptomyces/enzimologia , Streptomyces/genética , Especificidade por Substrato
5.
J Am Chem Soc ; 138(12): 4155-67, 2016 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-26982529

RESUMO

Modular polyketide synthases (PKSs) direct the biosynthesis of clinically valuable secondary metabolites in bacteria. The fidelity of chain growth depends on specific recognition between successive subunits in each assembly line: interactions mediated by C- and N-terminal "docking domains" (DDs). We have identified a new family of DDs in trans-acyl transferase PKSs, exemplified by a matched pair from the virginiamycin (Vir) system. In the absence of C-terminal partner (VirA (C)DD) or a downstream catalytic domain, the N-terminal DD (VirFG (N)DD) exhibits multiple characteristics of an intrinsically disordered protein. Fusion of the two docking domains results in a stable fold for VirFG (N)DD and an overall protein-protein complex of unique topology whose structure we support by site-directed mutagenesis. Furthermore, using small-angle X-ray scattering (SAXS), the positions of the flanking acyl carrier protein and ketosynthase domains have been identified, allowing modeling of the complete intersubunit interface.


Assuntos
Aciltransferases/metabolismo , Policetídeo Sintases/metabolismo , Virginiamicina/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Virginiamicina/metabolismo
6.
Dev Comp Immunol ; 53(2): 371-84, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26277106

RESUMO

Urodele amphibians are an interesting model because although they possess the cardinal elements of the vertebrate immune system, their immune response is apparently subdued. This phenomenon, sometimes regarded as a state of immunodeficiency, has been attributed by some authors to limited antibody diversity. We reinvestigated this issue in Pleurodeles waltl, a metamorphosing urodele, and noted that upsilon transcripts of its IgY repertoire were as diverse as alpha transcripts of the mammalian IgA repertoire. Mu transcripts encoding the IgM repertoire were less diverse, but could confer more plasticity. Both isotypes present potential polyreactive features that may confer urodele antibodies with the ability to bind to a variety of antigens. Finally, we observed additional cysteines in CDR1 and 2 of the IGHV5 and IGHV6 domains, some of which specific to urodeles, that could allow the establishment of a disulfide bond between these CDRs. Together, these data suggest that urodele antibody diversity is not as low as previously thought.


Assuntos
Regiões Determinantes de Complementaridade/genética , Cisteína/genética , Cadeias Pesadas de Imunoglobulinas/genética , Pleurodeles/imunologia , Animais , Diversidade de Anticorpos , Epitopos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Imunoglobulina A/genética , Imunoglobulina M/genética , Estágios do Ciclo de Vida , Mamíferos , Pleurodeles/crescimento & desenvolvimento , Especificidade da Espécie
7.
Chem Biol Interact ; 202(1-3): 70-7, 2013 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-23237860

RESUMO

Until the last decade, two unrelated aldehyde dehydrogenase (ALDH) superfamilies, i.e. the phosphorylating and non-phosphorylating superfamilies, were known to catalyze the oxidation of aldehydes to activated or non-activated acids. However, a third one was discovered by the crystal structure of a bifunctional enzyme 4-hydroxy-2-ketovalerate aldolase/acylating acetaldehyde dehydrogenase (DmpFG) from Pseudomonas sp. strain CF600 (Manjasetty et al., Proc. Natl. Acad. Sci. USA 100 (2003) 6992-6997). Indeed, DmpF exhibits a non-phosphorylating CoA-dependent ALDH activity, but is structurally related to the phosphorylating superfamily. In this study, we undertook the characterization of the catalytic and structural properties of MhpEF from Escherichia coli, an ortholog of DmpFG in which MhpF converts acetaldehyde, produced by the cleavage of 4-hydroxy-2-ketovalerate by MhpE, into acetyl-CoA. The kinetic data obtained under steady-state and pre-steady-state conditions show that the aldehyde dehydrogenase, MhpF, is active as a monomer, a unique feature relative to the phosphorylating and non-phosphorylating ALDH superfamilies. Our results also reveal that the catalytic properties of MhpF are not dependent on its oligomeric state, supporting the hypothesis of a structurally and catalytically independent entity. Moreover, the transthioesterification is shown to be rate-limiting and, when compared with a chemical model, its catalytic efficiency is increased 10(4)-fold. Therefore, CoA binding to MhpF increases its reactivity and optimizes its positioning relative to the thioacylenzyme intermediate, thus enabling the formation of an efficient deacylation complex.


Assuntos
Aldeído Oxirredutases/química , Aldeído Oxirredutases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Coenzima A/química , Coenzima A/metabolismo , Aldeído Desidrogenase/química , Aldeído Desidrogenase/metabolismo , Catálise , Cristalização/métodos , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Cinética , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Difração de Raios X/métodos
8.
Biomol Concepts ; 3(2): 141-50, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25436528

RESUMO

Abstract Thioredoxins (Trx) are ubiquitous proteins that are conserved in all living organisms from archaea to humans. These small proteins display various cellular roles, including functioning as reductases in redox processes. All Trxs share a similar, characteristic three-dimensional fold with the Cys-Pro-Gly-Cys motif that contains both the catalytic and the resolving cysteine (Cys) on the surface of the protein. Reaction of reduced Trx with its oxidized protein partners leads to formation of a transient interdisulfide intermediate. However, the short lifetime of this species hinders the characterization of the stabilizing interactions that occur between the partners. In this short review, the three-dimensional structures of four artificial covalent Trx-protein partner complexes are analyzed. The data show that interprotein stabilization is mainly due to hydrophobic contacts and main-chain hydrogen bonds but that no common recognition motif between Trx and its protein partners can be identified. In two cases, formation of the Trx-partner complex is accompanied by a significant conformational change of the protein target, although in no case does the conformation of Trx change significantly. The absence of a common recognition motif supports the idea that it is difficult to predict with confidence putative oxidized protein substrates of Trx using only soft docking and molecular simulation methods. Instead, biochemical methods including proteomic approaches remain the primary tools to identify novel protein substrates of Trx. The generality and relevance of methods used to identify which of the two Cys of the disulfide-oxidized protein partner forms the transient interdisulfide intermediate with Trx are also discussed.

10.
J Biol Chem ; 285(32): 25033-43, 2010 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-20489204

RESUMO

A new family of methionine-sulfoxide reductase (Msr) was recently described. The enzyme, named fRMsr, selectively reduces the R isomer at the sulfoxide function of free methionine sulfoxide (Met-R-O). The fRMsrs belong to the GAF fold family. They represent the first GAF domain to show enzymatic activity. Two other Msr families, MsrA and MsrB, were already known. MsrA and MsrB reduce free Met-S-O and Met-R-O, respectively, but exhibit higher catalytic efficiency toward Met-O within a peptide or a protein context. The fold of the three families differs. In the present work, the crystal structure of the fRMsr from Neisseria meningitidis has been determined in complex with S-Met-R-O. Based on biochemical and kinetic data as well as genomic analyses, Cys(118) is demonstrated to be the catalytic Cys on which a sulfenic acid is formed. All of the structural factors involved in the stereoselectivity of the l-Met-R-O binding were identified and account for why Met-S-O, DMSO, and a Met-O within a peptide are not substrates. Taking into account the structural, enzymatic, and biochemical information, a scenario of the catalysis for the reductase step is proposed. Based on the thiol content before and after Met-O reduction and the stoichiometry of Met formed per subunit of wild type and Cys-to-Ala mutants, a scenario of the recycling process of the N. meningitidis fRMsr is proposed. All of the biochemical, enzymatic, and structural properties of the N. meningitidis fRMsr are compared with those of MsrA and MsrB and are discussed in terms of the evolution of function of the GAF domain.


Assuntos
Metionina Sulfóxido Redutases/química , Neisseria meningitidis/enzimologia , Alanina/química , Catálise , Cisteína/química , Dimerização , Dissulfetos/química , Genômica , Cinética , Modelos Químicos , Mutação , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Ácidos Sulfênicos/química
11.
J Virol ; 82(19): 9577-90, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18632861

RESUMO

The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-A resolution of active coxsackievirus B3 RdRp (also named 3D(pol)) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3D(pol) at this site excludes its uridylation by the carrier 3D(pol). We suggest that VPg at this position is either uridylated by another 3D(pol) molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3D(pol)/VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.


Assuntos
Enterovirus/enzimologia , Picornaviridae/enzimologia , RNA Replicase/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X/métodos , Difosfatos/química , Enterovirus/genética , Genoma Viral , Guanosina Trifosfato/química , Conformação Molecular , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Homologia de Sequência de Aminoácidos , Replicação Viral
12.
J Bacteriol ; 190(7): 2556-64, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18245280

RESUMO

The yfdXWUVE operon appears to encode proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions. Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, findings from structural genomic studies have shown that the structure of YfdW, the protein encoded by the yfdW gene, is homologous to that of the enzyme that mediates oxalate catabolism in the obligate anaerobe Oxalobacter formigenes, O. formigenes formyl coenzyme A transferase (FRC). We now report the first detailed examination of the steady-state kinetic behavior and substrate specificity of recombinant, wild-type YfdW. Our studies confirm that YfdW is a formyl coenzyme A (formyl-CoA) transferase, and YfdW appears to be more stringent than the corresponding enzyme (FRC) in Oxalobacter in employing formyl-CoA and oxalate as substrates. We also report the effects of replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the E. coli protein. The results of these experiments show that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Our findings illustrate the utility of structural homology in assigning enzyme function and raise the question of whether oxalate catabolism takes place in E. coli upon the up-regulation of the yfdXWUVE operon under acidic conditions.


Assuntos
Proteínas de Bactérias/metabolismo , Coenzima A-Transferases/metabolismo , Proteínas de Escherichia coli/metabolismo , Oxalobacter formigenes/enzimologia , Acil Coenzima A/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Coenzima A-Transferases/química , Coenzima A-Transferases/genética , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glutamina/genética , Glutamina/metabolismo , Cinética , Dados de Sequência Molecular , Estrutura Molecular , Oxalatos/metabolismo , Oxalobacter formigenes/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Triptofano/genética , Triptofano/metabolismo
13.
Artigo em Inglês | MEDLINE | ID: mdl-17554171

RESUMO

The Picornaviridae virus family contains a large number of human pathogens such as poliovirus, hepatitis A virus and rhinoviruses. Amongst the viruses belonging to the genus Enterovirus, several serotypes of coxsackievirus coexist for which neither vaccine nor therapy is available. Coxsackievirus B3 is involved in the development of acute myocarditis and dilated cardiomyopathy and is thought to be an important cause of sudden death in young adults. Here, the first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. Standard crystallization methods yielded crystals that were poorly suited to X-ray diffraction studies, with one axis being completely disordered. Crystallization was improved by testing crystallization solutions from commercial screens as additives. This approach yielded crystals that diffracted to 2.1 A resolution and that were suitable for structure determination.


Assuntos
Enterovirus Humano B/enzimologia , RNA Replicase/química , Cristalização , Enterovirus Humano B/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , RNA Replicase/biossíntese , RNA Replicase/genética , RNA Replicase/isolamento & purificação
14.
J Biol Chem ; 282(14): 10678-89, 2007 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-17287213

RESUMO

Viruses of the family Flaviviridae are important human and animal pathogens. Among them, the Flaviviruses dengue (DENV) and West Nile (WNV) cause regular outbreaks with fatal outcomes. The RNA-dependent RNA polymerase (RdRp) activity of the non-structural protein 5 (NS5) is a key activity for viral RNA replication. In this study, crystal structures of enzymatically active and inactive WNV RdRp domains were determined at 3.0- and 2.35-A resolution, respectively. The determined structures were shown to be mostly similar to the RdRps of the Flaviviridae members hepatitis C and bovine viral diarrhea virus, although with unique elements characteristic for the WNV RdRp. Using a reverse genetic system, residues involved in putative interactions between the RNA-cap methyltransferase (MTase) and the RdRp domain of Flavivirus NS5 were identified. This allowed us to propose a model for the structure of the full-length WNV NS5 by in silico docking of the WNV MTase domain (modeled from our previously determined structure of the DENV MTase domain) onto the RdRp domain. The Flavivirus RdRp domain structure determined here should facilitate both the design of anti-Flavivirus drugs and structure-function studies of the Flavivirus replication complex in which the multifunctional NS5 protein plays a central role.


Assuntos
RNA Replicase/química , Proteínas não Estruturais Virais/química , Vírus do Nilo Ocidental/enzimologia , Animais , Cristalografia por Raios X , Vírus da Diarreia Viral Bovina/química , Vírus da Diarreia Viral Bovina/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Hepacivirus/química , Hepacivirus/enzimologia , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , RNA Replicase/antagonistas & inibidores , RNA Replicase/metabolismo , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Febre do Nilo Ocidental/tratamento farmacológico , Febre do Nilo Ocidental/enzimologia , tRNA Metiltransferases/metabolismo
15.
J Virol ; 80(17): 8493-502, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16912299

RESUMO

Macro domains constitute a protein module family found associated with specific histones and proteins involved in chromatin metabolism. In addition, a small number of animal RNA viruses, such as corona- and toroviruses, alphaviruses, and hepatitis E virus, encode macro domains for which, however, structural and functional information is extremely limited. Here, we characterized the macro domains from hepatitis E virus, Semliki Forest virus, and severe acute respiratory syndrome coronavirus (SARS-CoV). The crystal structure of the SARS-CoV macro domain was determined at 1.8-Angstroms resolution in complex with ADP-ribose. Information derived from structural, mutational, and sequence analyses suggests a close phylogenetic and, most probably, functional relationship between viral and cellular macro domain homologs. The data revealed that viral macro domains have relatively poor ADP-ribose 1"-phosphohydrolase activities (which were previously proposed to be their biologically relevant function) but bind efficiently free and poly(ADP-ribose) polymerase 1-bound poly(ADP-ribose) in vitro. Collectively, these results suggest to further evaluate the role of viral macro domains in host response to viral infection.


Assuntos
Adenosina Difosfato Ribose/metabolismo , Vírus da Hepatite E/química , Histonas/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Vírus da SARS/química , Vírus da Floresta de Semliki/química , Adenosina Difosfato Ribose/análogos & derivados , Adenosina Difosfato Ribose/química , Sequência de Aminoácidos , Cristalografia , Vírus da Hepatite E/metabolismo , Histonas/química , Modelos Moleculares , Dados de Sequência Molecular , Poli(ADP-Ribose) Polimerases/metabolismo , Vírus da SARS/metabolismo , Vírus da Floresta de Semliki/metabolismo , Relação Estrutura-Atividade
16.
J Mol Biol ; 343(1): 29-41, 2004 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-15381418

RESUMO

In the context of a medium-scaled structural genomics program aiming at solving the structures of as many as possible bacterial unknown open reading frame products from Escherichia coli (Y prefix), we have solved the structure of YdcW at 2.1A resolution, using molecular replacement. According to its sequence identity, YdcW has been classified into the betaine aldehyde dehydrogenases family (EC 1.2.1.8), catalysing the oxidation of betaine aldehyde into glycine betaine. The structure of YdcW resembles that of other aldehyde dehydrogenases: it is tetrameric and binds a NADH molecule in each monomer. The NADH molecules, bound in the active site by soaking, are revealed to be in the "hydrolysis position". Activities experiments demonstrate that YdcW is more active on medium-chains aldehyde than on betaine aldehyde. However, soaking of betaine into YdcW crystals revealed its presence in one of the subunits, in two positions, a putative resting position and a hydride transfer ready position. Analysis of kinetics data and of the active site shape suggest an optimum binding of n-alkyl aldehydes up to seven to eight carbon atoms, possibly followed by a bulky cyclic or aromatic group.


Assuntos
Aldeído Desidrogenase/genética , Betaína/análogos & derivados , Cristalografia por Raios X , Escherichia coli/enzimologia , Aldeído Desidrogenase/química , Aldeído Desidrogenase/isolamento & purificação , Aldeído Desidrogenase/metabolismo , Sequência de Aminoácidos , Betaína/química , Sítios de Ligação , Cálcio/química , Domínio Catalítico , Escherichia coli/genética , Cinética , Modelos Moleculares , NADP/metabolismo , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Especificidade por Substrato , Água/química
17.
J Biol Chem ; 279(6): 4459-64, 2004 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-14594955

RESUMO

Pheromone binding proteins (PBPs) are small helical proteins ( approximately 13-17 kDa) present in several sensory organs from moth and other insect species. They are involved in the transport of pheromones from the sensillar lymph to the olfactory receptors. We report here the crystal structure of a PBP (Amel-ASP1) originating from the honey-bee (Apis mellifera) antennae and expressed as recombinant protein in the yeast Pichia pastoris. Crystals of Amel-ASP1 were obtained at pH 5.5 using the nano-drops technique of crystallization with a novel optimization procedure, and the structure was solved initially with the single-wavelength anomalous diffraction technique using sulfur anomalous dispersion. The structure of Amel-ASP1 has been refined at 1.6-A resolution. Its fold is roughly similar to that of other PBP/odorant binding proteins, presenting six helices and three disulfide bridges. Contrary to the PBPs from Bombyx mori (Sandler, B. H., Nikonova, L., Leal, W. S., and Clardy, J. (2000) Chem. Biol. 7, 143-151) and Leucophea maderae (Lartigue, A., Gruez, A., Spinelli, S., Riviere, S., Brossut, R., Tegoni, M., and Cambillau, C. (2003) J. Biol. Chem. 278, 30213-30218), the extended C terminus folds into the protein and forms a wall of the internal hydrophobic cavity. Its backbone groups establish two hydrogen bonds with a serendipitous ligand, n-butyl-benzene-sulfonamide, an additive used in plastics. This mode of binding might, however, mimic that used by one of the pheromonal blend components and illustrates the binding versatility of PBPs.


Assuntos
Proteínas de Transporte/química , Proteínas de Insetos/química , Animais , Abelhas/química , Abelhas/genética , Proteínas de Transporte/genética , Cristalização , Cristalografia por Raios X , Proteínas de Insetos/genética , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Eletricidade Estática , Enxofre/química
18.
J Biol Chem ; 278(47): 46974-82, 2003 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-12960167

RESUMO

C1q is a versatile recognition protein that binds to an amazing variety of immune and non-immune ligands and triggers activation of the classical pathway of complement. The crystal structure of the C1q globular domain responsible for its recognition properties has now been solved and refined to 1.9 A of resolution. The structure reveals a compact, almost spherical heterotrimeric assembly held together mainly by non-polar interactions, with a Ca2+ ion bound at the top. The heterotrimeric assembly of the C1q globular domain appears to be a key factor of the versatile recognition properties of this protein. Plausible three-dimensional models of the C1q globular domain in complex with two of its physiological ligands, C-reactive protein and IgG, are proposed, highlighting two of the possible recognition modes of C1q. The C1q/human IgG1 model suggests a critical role for the hinge region of IgG and for the relative orientation of its Fab domain in C1q binding.


Assuntos
Complemento C1q/química , Subunidades Proteicas/química , Proteína C-Reativa/química , Proteína C-Reativa/metabolismo , Cálcio/química , Complemento C1q/metabolismo , Cristalização , Cristalografia por Raios X , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Ligantes , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo
19.
J Biol Chem ; 278(36): 34582-6, 2003 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-12844490

RESUMO

Because of its toxicity, oxalate accumulation from amino acid catabolism leads to acute disorders in mammals. Gut microflora are therefore pivotal in maintaining a safe intestinal oxalate balance through oxalate degradation. Oxalate catabolism was first identified in Oxalobacter formigenes, a specialized, strictly anaerobic bacterium. Oxalate degradation was found to be performed successively by two enzymes, a formyl-CoA transferase (frc) and an oxalate decarboxylase (oxc). These two genes are present in several bacterial genomes including that of Escherichia coli. The frc ortholog in E. coli is yfdW, with which it shares 61% sequence identity. We have expressed the YfdW open reading frame product and solved its crystal structure in the apo-form and in complex with acetyl-CoA and with a mixture of acetyl-CoA and oxalate. YfdW exhibits a novel and spectacular fold in which two monomers assemble as interlaced rings, defining the CoA binding site at their interface. From the structure of the complex with acetyl-CoA and oxalate, we propose a putative formyl/oxalate transfer mechanism involving the conserved catalytic residue Asp169. The similarity of yfdW with bacterial orthologs (approximately 60% identity) and paralogs (approximately 20-30% identity) suggests that this new fold and parts of the CoA transfer mechanism are likely to be the hallmarks of a wide family of CoA transferases.


Assuntos
Coenzima A-Transferases/química , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Acetilcoenzima A/química , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Coenzima A-Transferases/metabolismo , Cristalografia por Raios X , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano , Modelos Moleculares , Dados de Sequência Molecular , Fases de Leitura Aberta , Oxalatos/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
20.
Acta Crystallogr D Biol Crystallogr ; 59(Pt 5): 919-21, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12777812

RESUMO

Pheromone-binding proteins (PBPs) are small helical proteins ( approximately 13-17 kDa) present in various sensory organs from moths and other insect species. They are involved in the transport of pheromones from the sensillar lymph to the olfactory receptors. Here, crystals of a PBP (Amel-ASP1) originating from honeybee (Apis mellifera L.) antennae and expressed as recombinant protein using the yeast Pichia pastoris are reported. Crystals of Amel-ASP1 have been obtained by the sitting-drop vapour-diffusion method using a nanodrop-dispensing robot under the following conditions: 200 nl of 40 mg ml(-1) protein solution in 10 mM Tris, 25 mM NaCl pH 8.0 was mixed with 100 nl of well solution containing 0.15 M sodium citrate, 1.5 M ammonium sulfate pH 5.5. The protein crystallizes in space group C222(1), with unit-cell parameters a = 74.8, b = 85.8, c = 50.2 A. With one molecule in the asymmetric unit, V(M) is 3.05 A(3) Da(-1) and the solvent content is 60%. A complete data set has been collected at 1.6 A resolution on beamline ID14-2 (ESRF, Grenoble). The nanodrop crystallization technique used with a novel optimization procedure made it possible to consume small amounts of protein and to obtain a unique crystal per nanodrop, suitable directly for data collection in-house or at a synchrotron-radiation source.


Assuntos
Abelhas/química , Proteínas de Transporte/química , Proteínas de Insetos , Sequência de Aminoácidos , Sulfato de Amônio/química , Animais , Proteínas de Transporte/genética , Cristalização/métodos , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Pichia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
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