RESUMO
In this chapter, we will focus on the role of bioinformatics to analyze a protein after its protein structure has been determined. First, we present how to validate protein structures for quality assurance. Then, we discuss how to analyze protein-protein interfaces and how to predict the biomolecule which is the biological oligomeric state of the protein. Finally, we discuss how to search for homologs based on the 3-D structure which is an essential step for understanding protein function.
Assuntos
Biologia Computacional , Proteínas/química , Bases de Dados de Proteínas , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/estatística & dados numéricos , Software , Homologia Estrutural de ProteínaAssuntos
Dioxigenases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalização , Bases de Dados de Proteínas , Dioxigenases/genética , Dioxigenases/metabolismo , Metais/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaAssuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ciclofilinas/química , Ciclofilinas/metabolismo , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Thermotoga maritima/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Peptidilprolil Isomerase/classificação , Polietilenoglicóis , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaAssuntos
Proteínas de Ligação a DNA/química , Thermotoga maritima/metabolismo , Sequência de Aminoácidos , Anisotropia , Cristalografia por Raios X , DNA de Cadeia Simples , Escherichia coli/metabolismo , Mitocôndrias/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Plasmídeos/metabolismo , Estrutura Secundária de Proteína , Fatores de Transcrição/química , Difração de Raios XRESUMO
Fosfomycin [(1R,2S)-epoxypropylphosphonic acid] is a simple phosphonate found to have antibacterial activity against both Gram-positive and Gram-negative microorganisms. Early resistance to the clinical use of the antibiotic was linked to a plasmid-encoded resistance protein, FosA, that catalyzes the addition of glutathione to the oxirane ring, rendering the antibiotic inactive. Subsequent studies led to the discovery of a genomically encoded homologue in the pathogen Pseudomonas aeruginosa. The proteins are Mn(II)-dependent enzymes where the metal is proposed to act as a Lewis acid stabilizing the negative charge that develops on the oxirane oxygen in the transition state. Several simple phosphonates, including the antiviral compound phosphonoformate (K(i) = 0.4 +/- 0.1 microM, K(d) approximately 0.2 microM), are shown to be inhibitors of FosA. The crystal structure of FosA from P. aeruginosa with phosphonoformate bound in the active site has been determined at 0.95 A resolution and reveals that the inhibitor forms a five-coordinate complex with the Mn(II) center with a geometry similar to that proposed for the transition state of the reaction. Binding studies show that phosphonoformate has a near-diffusion-controlled on rate (k(on) approximately 10(7)-10(8) M(-1) s(-1)) and an off rate (k(off) = 5 s(-1)) that is slower than that for fosfomycin (k(off) = 30 s(-1)). Taken together, these data suggest that the FosA-catalyzed reaction has a very early transition state and phosphonoformate acts as a minimal transition state analogue inhibitor.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Farmacorresistência Bacteriana , Foscarnet/química , Fosfomicina/química , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/química , Pseudomonas aeruginosa/química , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Catálise , Cristalização , Cristalografia por Raios X , Glutationa Transferase/isolamento & purificação , Cinética , Manganês/química , Organofosfonatos/químicaRESUMO
The crystal structure of fosfomycin resistance protein FosA from transposon Tn2921 has been established at a resolution of 2.5 A. The protein crystallized without bound Mn(II) and K+, ions crucial for efficient catalysis, providing a structure of the apo enzyme. The protein maintains the three-dimensional domain-swapped arrangement of the paired betaalphabetabetabeta-motifs observed in the genomically encoded homologous enzyme from Pseudomonas aeruginosa (PA1129). The basic architecture of the active site is also maintained, despite the absence of the catalytically essential Mn(II). However, the absence of K+, which has been shown to enhance enzymatic activity, appears to contribute to conformational heterogeneity in the K(+)-binding loops.
Assuntos
Proteínas de Bactérias/química , Glutationa Transferase/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Elementos de DNA Transponíveis/genética , Dimerização , Farmacorresistência Bacteriana/genética , Fosfomicina/química , Fosfomicina/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Manganês/química , Dados de Sequência Molecular , Potássio/química , Conformação Proteica , Pseudomonas aeruginosa/genéticaRESUMO
The class kappa glutathione (GSH) transferase is an enzyme that resides in the mitochondrial matrix. Its relationship to members of the canonical GSH transferase superfamily has remained an enigma. The three-dimensional structure of the class kappa enzyme from rat (rGSTK1-1) in complex with GSH has been solved by single isomorphous replacement with anomalous scattering at a resolution of 2.5 A. The structure reveals that the enzyme is more closely related to the protein disulfide bond isomerase, dsbA, from Escherichia coli than it is to members of the canonical superfamily. The structures of rGSTK1-1 and the canonical superfamily members indicate that the proteins folds have diverged from a common thioredoxin/glutaredoxin progenitor but did so by different mechanisms. The mitochondrial enzyme, therefore, represents a fourth protein superfamily that supports GSH transferase activity. The thioredoxin domain functions in a manner that is similar to that seen in the canonical enzymes by providing key structural elements for the recognition of GSH. The hydroxyl group of S16 is within hydrogen-bonding distance of the sulfur of bound GSH and is, in part, responsible for the ionization of the thiol in the E*GSH complex (pKa = 6.4 +/- 0.1). Preequilibrium kinetic experiments indicate that the k(on) for GSH is 1 x 10(5) M(-1) s(-1) and k(off) for GS- is approximately 8 s(-1) and relatively slow with respect to turnover with 1-chloro-2, 4-dinitrobenzene (CDNB). As a result, the KM(GSH) (11 mM) is much larger than the apparent Kd(GSH) (90 microM). The active site has a relatively open access channel that is flanked by disordered loops that may explain the relatively high turnover number (280 s(-1) at pH 7.0) toward CDNB. The disordered loops form an extensive contiguous patch on one face of the dimeric enzyme, a fact that suggests that the protein surface may interact with a membrane or other protein partner.
Assuntos
Evolução Molecular , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Mitocôndrias/enzimologia , Substituição de Aminoácidos/genética , Animais , Sítios de Ligação , Catálise , Cristalografia por Raios X , Dimerização , Glutationa/química , Glutationa/metabolismo , Glutationa Transferase/genética , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Serina/genética , Relação Estrutura-AtividadeRESUMO
Multiple sequence alignments of the eight glutathione (GSH) transferase homologues encoded in the genome of Escherichia coli were used to define a consensus sequence for the proteins. The consensus sequence was analyzed in the context of the three-dimensional structure of the gst gene product (EGST) obtained from two different crystal forms of the enzyme. The enzyme consists of two domains. The N-terminal region (domain I) has a thioredoxin-like alpha/beta-fold, while the C-terminal domain (domain II) is all alpha-helical. The majority of the consensus residues (12/17) reside in the N-terminal domain. Fifteen of the 17 residues are involved in hydrophobic core interactions, turns, or electrostatic interactions between the two domains. The results suggest that all of the homologues retain a well-defined group of structural elements both in and between the N-terminal alpha/beta domain and the C-terminal domain. The conservation of two key residues for the recognition motif for the gamma-glutamyl-portion of GSH indicates that the homologues may interact with GSH or GSH analogues such as glutathionylspermidine or alpha-amino acids. The genome context of two of the homologues forms the basis for a hypothesis that the b2989 and yibF gene products are involved in glutathionylspermidine and selenium biochemistry, respectively.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Genoma Bacteriano , Sequência de Aminoácidos , Sítios de Ligação/genética , Sequência Conservada/genética , Cristalografia por Raios X , Glutationa Transferase/química , Glutationa Transferase/genética , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The fosfomycin resistance protein (FosA) catalyzes the Mn(II)- and K+-dependent addition of glutathione to the oxirane of the antibiotic fosfomycin. The crystal structure of FosA from Pseudomonas aeruginosa was solved at a resolution of 1.19 A by multiwavelength anomalous diffraction at the L-III edge of a Tl+ derivative. The structure solution took advantage of the ability of Tl+ to substitute for K+. The existence of multiple Tl sites in the asymmetric unit suggests that this may be a generally useful technique for phasing protein crystal structures. A 1.35 A resolution structure with phosphate bound in the active site shows that the Mn(II) center has a rare four-coordinate geometry. The structure of the fosfomycin complex at 1.19 A resolution indicates that the Mn(II) center is close to five-coordinate with trigonal bipyramidal geometry and a ligand set consisting of two histidines (H7 and H64) and one phosphonate oxygen occupying the equatorial sites and the carboxylate of E110 at one of the apical sites. The oxirane oxygen of the substrate is located at the other apical site but is 0.2 A beyond the average Mn-O distance for five-coordinate Mn(II). The Mn(II) center is proposed to stabilize the alkoxide in the transition state, while the nearby hydroxyl group of T9 acts as a proton donor in the reaction. The K+ ion located 6.5 A from the Mn(II) appears to help orient the substrate for nucleophilic attack.
Assuntos
Proteínas de Bactérias , Glutationa Transferase/química , Tálio/química , Sítios de Ligação , Catálise , Cristalografia por Raios X , Glutationa Transferase/genética , Metaloproteínas/química , Metaloproteínas/genética , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genéticaRESUMO
FosA is a manganese metalloglutathione transferase that confers resistance to the broad-spectrum antibiotic fosfomycin, (1R,2S)-epoxypropylphosphonic acid. The reaction catalyzed by FosA involves the attack by glutathione on fosfomycin to yield the product 1-(S-glutathionyl)-2-hydroxypropylphosphonic acid. The enzyme is a dimer of 16 kDa subunits, each of which harbors one mononuclear Mn(II) site. The coordination environment of the Mn(II) in the FosA x Mn(2+) complex is composed of a glutamate and two histidine ligands and three water molecules. Here we report EPR spectroscopic studies on FosA, in which EPR spectra were obtained at 35 GHz and 2 K using dispersion-detection rapid-passage techniques. This approach provides an absorption envelope line shape, in contrast to the conventional (slow-passage) derivative line shape, and is a more reliable way to collect spectra from Mn(II) centers with large zero-field splitting. We obtain excellent spectra of FosA bound with substrate, substrate analogue phosphate ion, and product, whereas these states cannot be studied by X-band, slow-passage methods. Simulation of the EPR spectra shows that binding of substrate or analogue causes a profound change in the electronic parameters of the Mn(II) ion. The axial zero-field splitting changes from [D] = 0.06 cm(-1) for substrate-free enzyme to 0.23 cm(-1) for fosfomycin-bound enzyme, 0.28 (1) cm(-1) for FosA with phosphate, and 0.27 (1) cm(-1) with product. Such a large zero-field splitting is uncommon for Mn(II). A simple ligand field analysis of this change indicates that binding of the phosphonate/phosphate group of substrate or analogue changes the electronic energy levels of the Mn(II) 3d orbitals by several thousand cm(-1), indicative of a significant change in the Mn(II) coordination sphere. Comparison with related enzymes (glyoxalase I and MnSOD) suggests that the change in the coordination environment on substrate binding may correspond to loss of the glutamate ligand.
