RESUMEN
Human glucose-6-phosphate dehydrogenase (G6PD) is NADP(+)-dependent and catalyses the first and rate-limiting step of the pentose phosphate shunt. Binary complexes of the human deletion mutant, DeltaG6PD, with glucose-6-phosphate and NADP(+) have been crystallized and their structures solved to 2.9 and 2.5 A, respectively. The structures are compared with the previously determined structure of the Canton variant of human G6PD (G6PD(Canton)) in which NADP(+) is bound at the structural site. Substrate binding in DeltaG6PD is shown to be very similar to that described previously in Leuconostoc mesenteroides G6PD. NADP(+) binding at the coenzyme site is seen to be comparable to NADP(+) binding in L. mesenteroides G6PD, although some differences arise as a result of sequence changes. The tetramer interface varies slightly among the human G6PD complexes, suggesting flexibility in the predominantly hydrophilic dimer-dimer interactions. In both complexes, Pro172 of the conserved peptide EKPxG is in the cis conformation; it is seen to be crucial for close approach of the substrate and coenzyme during the enzymatic reaction. Structural NADP(+) binds in a very similar way in the DeltaG6PD-NADP(+) complex and in G6PD(Canton), while in the substrate complex the structural NADP(+) has low occupancy and the C-terminal tail at the structural NADP(+) site is disordered. The implications of possible interaction between the structural NADP(+) and G6P are considered.
Asunto(s)
Glucosa-6-Fosfato/metabolismo , Glucosafosfato Deshidrogenasa/metabolismo , NADP/metabolismo , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Interpretación Estadística de Datos , Escherichia coli/química , Escherichia coli/genética , Glucosa-6-Fosfato/química , Glucosafosfato Deshidrogenasa/química , Glucosafosfato Deshidrogenasa/genética , Humanos , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Mutación/fisiología , NADP/química , Unión Proteica , Conformación ProteicaRESUMEN
The neisserial surface protein A (NspA) from Neisseria meningitidis is a promising vaccine candidate because it is highly conserved among meningococcal strains and induces bactericidal antibodies. NspA is a homolog of the Opa proteins, which mediate adhesion to host cells. Here, we present the crystal structure of NspA, determined to 2.55-A resolution. NspA forms an eight-stranded antiparallel beta-barrel. The four loops at the extracellular side of the NspA molecule form a long cleft, which contains mainly hydrophobic residues and harbors a detergent molecule, suggesting that the protein might function in the binding of hydrophobic ligands, such as lipids. In addition, the structure provides a starting point for structure-based vaccine design.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Vacunas Bacterianas/química , Neisseria meningitidis/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Diseño de Fármacos , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
Proteases perform a wide variety of functions, inside and outside cells, regulating many biological processes. Recent years have witnessed a number of significant advances in the structural biology of proteases, including aspects of intracellular protein and peptide degradation by self-compartmentalizing proteases, activation of proteases in proteolytic cascades of regulatory pathways, and mechanisms of microbial proteases in pathogenicity.
Asunto(s)
Endopeptidasas/metabolismo , Estructura Terciaria de Proteína , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Endopeptidasas/química , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
OmpT is an integral outer membrane protease of Escherichia coli. Overexpression of OmpT in E. coli and subsequent in vitro folding of the produced inclusion bodies yielded protein with a native-like structure. However, enzymatically active protease was only obtained after addition of the outer membrane lipid lipopolysaccharide (LPS). OmpT is the first example of an enzyme that requires LPS for activity. In this study, we investigated the nature of this activation. Circular dichroism analysis showed that binding of LPS did not lead to large structural changes. Titration of OmpT with LPS and determining the resulting OmpT activity with a fluorimetric assay yielded a dissociation constant of 10-4 m for E. coli K-12 LPS. Determining the dissociation constants for different LPS chemotypes revealed that a fully acylated lipid A part is minimally required for activation of OmpT. The heptose-bound phosphates in the inner core region were also important for activation. The affinity for LPS was not dependent on the concentration of substrate, neither was affinity for the substrate influenced by the concentration of LPS. This indicated that LPS most likely does not act at the level of substrate binding. We hypothesize that LPS induces a subtle conformational change in the protein that is required for obtaining a native active site geometry.