RESUMO
The structure of the nucleotide-binding domain of the Mg-ATPase MgtA from Escherichia coli has been solved and refined to 1.6 A resolution. The structure is made up of a six-stranded beta-sheet and a bundle of three alpha-helices, with the nucleotide-binding site sandwiched in between. The MgtA nucleotide-binding domain is shorter and more compact compared with that of the related Ca-ATPase and lacks one of the beta-strands at the edge of the beta-sheet. The ATP-binding pocket is surrounded by three sequence and structural motifs known from other P-type ATPases and a fourth unique motif that is found only in Mg-ATPases. This motif consists of a short polypeptide stretch running very close to the ATP-binding site, while in Ca-ATPase the binding site is more open, with the corresponding polypeptide segment folded away from the active site.
Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras/química , Domínios e Motivos de Interação entre Proteínas , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Understanding of how P-type ATPases work would greatly benefit from the elucidation of more high-resolution structures. The nucleotide-binding domain of Mg-ATPase was selected for structural studies because Mg-ATPase is closely related to eukaryotic Ca-ATPase and Na,K-ATPase while the nucleotide-binding domain itself has diverged substantially. Two fragments of Mg-ATPase were cloned in Escherichia coli and purified. The entire cytoplasmic loop (residues 367-673), consisting of the phosphorylation and nucleotide-binding domains, expressed well and was purified in large quantities. The smaller 19.5 kDa nucleotide-binding domain (residues 383-545) expressed less well but formed crystals that diffracted to a resolution of 1.53 A which will be used for molecular replacement.
Assuntos
Adenosina Trifosfatases/química , ATPase de Ca(2+) e Mg(2+)/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras/química , Nucleotídeos/metabolismo , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
Glutaredoxin Grx1p from yeast was crystallized both as an independent protein and in a protein fusion with His-tagged yellow fluorescent protein (rxYFP). A glutathionylated C30S mutant of the 12 kDa Grx1p was crystallized in two different forms in PEG 4000 at low pH. These orthorhombic and monoclinic forms diffract to 2.0 A (synchrotron radiation) and 2.7 A (rotating-anode generator), respectively. In contrast, rxYFP-Grx1p formed crystals at high pH in MgSO(4) which diffract synchrotron radiation to 2.7 A.
Assuntos
Mutação , Oxirredutases/química , Oxirredutases/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalização , Cristalografia por Raios X , Glutarredoxinas , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
The structure of the N-domain of porcine alpha(2) Na,K-ATPase was determined crystallographically to 3.2A resolution by isomorphous heavy-atom replacement using a single mercury derivative. The structure was finally refined against 2.6A resolution synchrotron data. The domain forms a seven-stranded antiparallel beta-sheet with two additional beta-strands forming a hairpin and five alpha-helices. Approximately 75% of the residues were superimposable with residues from the structure of Ca-ATPase N-domain, and a structure-based sequence alignment is presented. The positions of key residues are discussed in relation to the pattern of hydrophobicity, charge and sequence conservation of the molecular surface. The structure of a hexahistidine tag binding to nickel ions is presented.
Assuntos
ATPase Trocadora de Sódio-Potássio/química , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Cristalografia por Raios X , Íons , Mercúrio/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Suínos , SíncrotronsRESUMO
The ectodomain of the matrix 2 protein (M2e) of influenza A virus represents an attractive target for developing a universal influenza A vaccine, with its sequence being highly conserved amongst human variants of this virus. With the aim of targeting conformational epitopes presumably shared by diverse influenza A viruses, a vaccine (M2e-NSP4) was constructed linking M2e (in its consensus sequence) to the rotavirus fragment NSP4(98-135); due to its coiled-coil region this fragment is known to form tetramers in aqueous solution and in this manner we hoped to mimick the natural configuration of M2e as presented in membranes. M2e-NSP4 was then evaluated side-by-side with synthetic M2e peptide for its immunogenicity and protective efficacy in a murine influenza challenge model. Here we demonstrate that M2e fused to the tetramerizing protein induces an accelerated, augmented and more broadly reactive antibody response than does M2e peptide as measured in two different assays. Most importantly, vaccination with M2e-NSP4 caused a significant decrease in lung virus load early after challenge with influenza A virus and maintained its efficacy against a lethal challenge even at very low vaccine doses. Based on the results presented in this study M2e-NSP4 merits further investigation as a candidate for or as a component of a universal influenza A vaccine.
Assuntos
Glicoproteínas/imunologia , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/imunologia , Toxinas Biológicas/imunologia , Proteínas da Matriz Viral/imunologia , Proteínas não Estruturais Virais/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Cromatografia de Afinidade , Clonagem Molecular , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Glicoproteínas/genética , Células HeLa , Humanos , Vacinas contra Influenza/genética , Vacinas contra Influenza/farmacologia , Estimativa de Kaplan-Meier , Pulmão/efeitos dos fármacos , Pulmão/virologia , Camundongos , Dados de Sequência Molecular , Infecções por Orthomyxoviridae/prevenção & controle , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Toxinas Biológicas/genética , Carga Viral/imunologia , Proteínas da Matriz Viral/genética , Proteínas não Estruturais Virais/genéticaRESUMO
Glutathionylated glutaredoxin Grx1p C30S mutant from yeast has been crystallized in space group C222(1) and a fusion protein between redox-sensitive yellow fluorescent protein (rxYFP) and Grx1p C30S has been crystallized in space group P6(4). The structure of the latter was solved by molecular replacement using the known rxYFP structure as a search model. The structure of the Grx1p moiety was built and the structure was refined against 2.7 A synchrotron data to an R(free) of 25.7%. There are no specific contacts between the two domains, indicating that the observed enhanced exchange of reduction equivalents between them arises from diffusion or from an enhanced collision rate in solution. The Grx1p structure thus obtained was subsequently used to solve the structure of the orthorhombic crystal, which could be refined against 2.0 A data to an R(free) of 24.3%. The structure of the glutathione-bound protein and the glutaredoxin domain in the fusion protein are similar. The covalent disulfide bond between the glutathione and protein is broken upon exposure to synchrotron radiation. The structure and the glutathione-binding mode are described and compared with existing crystallographic and nuclear magnetic resonance (NMR) structures of related glutaredoxins. Conserved residues are clustered on one side of the active site.
Assuntos
Oxirredutases/química , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Glutarredoxinas , Glutationa/genética , Glutationa/metabolismo , Proteínas Luminescentes/genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Oxirredutases/genética , Oxirredutases/metabolismo , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Enterococcus faecalis haem catalase was crystallized using lithium sulfate at neutral pH. The crystals belong to space group R3, with unit-cell parameters a = b = 236.9, c = 198.1 A. The three-dimensional structure was determined by molecular replacement using a subunit of the Proteus mirabilis catalase structure. It was refined against 2.3 A synchrotron data to a free R factor of 21.8%. Like other catalases, the E. faecalis catalase is a homotetramer with a fold and structure similar to those of its structurally closest relative P. mirabilis. The solvent structure in the active site is identical in the four subunits but differs from that found in other catalases. The structural consequences of the Ramachandran outlier Ser196 are discussed.
Assuntos
Catalase/química , Enterococcus faecalis/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Catalase/metabolismo , Cristalografia por Raios X , Heme/química , Heme/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Serina/metabolismo , Solventes/químicaRESUMO
The nucleotide-binding domain of the Na,K-ATPase ion pump was expressed with a His tag in Escherichia coli and purified. The soluble 24 kDa derivative consists of 214 amino-acid residues and was crystallized in the presence of NiCl(2). The crystals belong to space group F23, with unit-cell parameters a = b = c = 147.5 A, and diffract to 3.1 A. Complete data sets could be collected from native and thimerosal-treated crystals frozen in 50% sucrose. Five mercury positions were found and initial SIR phases calculated.
Assuntos
ATPase Trocadora de Sódio-Potássio/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Cristalização/métodos , Proteínas de Membrana/química , Níquel , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Suínos , Timerosal , Difração de Raios X/métodosRESUMO
The cell membrane Na,K-ATPase is a member of the P-type family of active cation transport proteins. Recently the molecular structure of the related sarcoplasmic reticulum Ca-ATPase in an E1 conformation has been determined at 2.6 A resolution. Furthermore, theoretical models of the Ca-ATPase in E2 conformations are available. As a result of these developments, these structural data have allowed construction of homology models that address the central questions of mechanism of active cation transport by all P-type cation pumps. This review relates recent evidence on functional sites of Na,K-ATPase for the substrate (ATP), the essential cofactor (Mg(2+) ions), and the transported cations (Na(+) and K(+)) to the molecular structure. The essential elements of the Ca-ATPase structure, including 10 transmembrane helices and well-defined N, P, and A cytoplasmic domains, are common to all PII-type pumps such as Na,K-ATPase and H,K-ATPases. However, for Na,K-ATPase and H,K-ATPase, which consist of both alpha- and beta-subunits, there may be some detailed differences in regions of subunit interactions. Mutagenesis, proteolytic cleavage, and transition metal-catalyzed oxidative cleavages are providing much evidence about residues involved in binding of Na(+), K(+), ATP, and Mg(2+) ions and changes accompanying E1-E2 or E1-P-E2-P conformational transitions. We discuss this evidence in relation to N, P, and A cytoplasmic domain interactions, and long-range interactions between the active site and the Na(+) and K(+) sites in the transmembrane segments, for the different steps of the catalytic cycle.