RESUMEN
The ubiquitous Hsp90 chaperone participates in snoRNP and RNA polymerase assembly through interaction with the R2TP complex. This complex includes the proteins Tah1, Pih1, Rvb1, and Rvb2. Tah1 bridges Hsp90 to R2TP. Its minimal TPR domain includes two TPR motifs and a capping helix. We established the high-resolution solution structures of Tah1 free and in complex with the Hsp90 C-terminal peptide. The TPR fold is similar in the free and bound forms and we show experimentally that in addition to its solvating/stabilizing role, the capping helix is essential for the recognition of the Hsp90 (704)EMEEVD(709) motif. In addition to Lys79 and Arg83 from the carboxylate clamp, this helix bears Tyr82 forming a π/S-CH3 interaction with Hsp90 M(705) from the peptide 310 helix. The Tah1 C-terminal region is unfolded, and we demonstrate that it is essential for the recruitment of the Pih1 C-terminal domain and folds upon binding.
Asunto(s)
Proteínas HSP90 de Choque Térmico/química , Chaperonas Moleculares/química , Proteínas Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae , Secuencia de Aminoácidos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Técnicas del Sistema de Dos HíbridosRESUMEN
The DsbD protein is essential for electron transfer from the cytoplasm to the periplasm of Gram-negative bacteria. Its N-terminal domain dispatches electrons coming from cytoplasmic thioredoxin (Trx), via its central transmembrane and C-terminal domains, to its periplasmic partners: DsbC, DsbE/CcmG, and DsbG. Previous structural studies described the latter proteins as Trx-like folds possessing a characteristic C-X-X-C motif able to generate a disulfide bond upon oxidation. The Escherichia coli nDsbD displays an immunoglobulin-like fold in which two cysteine residues (Cys103 and Cys109) allow a disulfide bond exchange with its biological partners.We have determined the structure in solution and the backbone dynamics of the C103S mutant of the N-terminal domain of DsbD from Neisseria meningitidis. Our results highlight significant structural changes concerning the beta-sheets and the local topology of the active site compared with the oxidized form of the E. coli nDsbD. The structure reveals a "cap loop" covering the active site, similar to the oxidized E. coli nDsbD X-ray structure. However, regions featuring enhanced mobility were observed both near to and distant from the active site, revealing a capacity of structural adjustments in the active site and in putative interaction areas with nDsbD biological partners. Results are discussed in terms of functional consequences.
Asunto(s)
Cisteína/genética , Proteínas Mutantes/química , Mutación/genética , Neisseria meningitidis/enzimología , Oxidorreductasas/química , Oxidorreductasas/genética , Serina/genética , Dominio Catalítico , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Proteínas Mutantes/genética , Estructura Terciaria de Proteína/genética , SolucionesRESUMEN
The secreted form of the PilB protein was proposed to be involved in pathogen survival fighting against the defensive host's oxidative burst. PilB protein is composed of three domains. The central and the C-terminal domains display methionine sulfoxide reductase A and B activities, respectively. The N-terminal domain, which possesses a CXXC motif, was recently shown to regenerate in vitro the reduced forms of the methionine sulfoxide reductase domains of PilB from their oxidized forms, as does the thioredoxin 1 from E. coli, via a disulfide bond exchange. The thioredoxin-like N-terminal domain belongs to the cytochrome maturation protein structural family, but it possesses a unique additional segment (99)FLHE (102) localized in a loop. This segment covers one edge of the active site in the crystal structure of the reduced form of the N-terminal domain of PilB. We have determined the solution structure and the dynamics of the N-terminal domain from Neisseria meningitidis, in its reduced and oxidized forms. The FLHE loop adopts, in both redox states, a well-defined conformation. Subtle conformational and dynamic changes upon oxidation are highlighted around the active site, as well as in the FLHE loop. The functional consequences of the cytochrome maturation protein topology and those of the presence of FLHE loop are discussed in relation to the enzymatic properties of the N-terminal domain.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Neisseria meningitidis/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Oxidación-Reducción , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
We report the nearly complete 1H, 13C, and 15N resonance assignments of the C103S mutant of the N-terminal domain of DsbD from Neisseria meningitides. Secondary structure determination using CSI method leads to the prediction of nine beta-sheet parts.
Asunto(s)
Antígenos Bacterianos/química , Proteínas Bacterianas/química , Espectroscopía de Resonancia Magnética/métodos , Neisseria meningitidis/metabolismo , Secuencia de Aminoácidos , Isótopos de Carbono/química , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Neisseria meningitidis/genética , Isótopos de Nitrógeno/química , ProtonesRESUMEN
We report the nearly complete 1H, 13C and 15N resonance assignments of the oxidized form (Cys(67)-Cys(70)) of the N-terminal domain of PilB from Neisseria meningitidis. Secondary structure determination using CSI method and TALOS leads mainly to the prediction of 7 alpha-helical and 5 beta-sheet parts.
Asunto(s)
Proteínas Bacterianas/química , Neisseria meningitidis/química , Oxidorreductasas/química , Proteínas Bacterianas/genética , Cistina/química , Estructura Molecular , Neisseria meningitidis/genética , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Oxidorreductasas/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Aiming at contributing to the development of potential atheroprotective agents, we report on the concept and design of two peptide models, which mimic the amphipathic helices of apoA-I and incorporate Met into their sequences to validate its role as oxidant scavenger: Ac-ESK(Palm)KELSKSW(10)SEM(13)LKEK(Palm)SKS-NH(2) (model 1 [W(10), M(13)]) and Ac-ESK(Palm)KELSKSM(10)SEW(13)LKEK(Palm)SKS-NH(2) (model 2 [M(10), W(13)]). Hydrophobic residues of both models cover about the half of the surface, while the positively and negatively charged residues constitute two separate clusters on the hydrophilic face. Palmitoyl groups were introduced into the Lys-N(epsilon)H(2) groups at positions 3 and 17 to contribute to the amphipathic character of the peptides and stabilize the nonpolar face of the helix. Conformational study by the combined application of 2D-NMR and molecular dynamics simulations, CD, FTIR, and fluorescence spectroscopy revealed that model 1 adopts helical conformation and Met is well exposed to the microenvironment. Model 2 that derives from model 1 by exchanging W(10) (model 1) with M(10) and M(13) (model 1) with W(13) also displays helical characteristics, while Met is rather shielded. Oxidation experiments indicated that model 1 exhibits a 2-fold more potent antioxidant activity towards LDL oxidation, compared to model 2, confirming the role of Met, when is devoid of steric hindrances, as oxidant scavenger for the protection of LDL.