RESUMEN
Two previously reported holoprotein crystal forms of the flavodoxin-like E. coli protein WrbA, diffracting to 2.6 and 2.0 A resolution, and new crystals of WrbA apoprotein diffracting to 1.85 A, are refined and analysed comparatively through the lens of flavodoxin structures. The results indicate that differences between apo- and holoWrbA crystal structures are manifested on many levels of protein organization as well as in the FMN-binding sites. Evaluation of the influence of crystal contacts by comparison of lattice packing reveals the protein's global response to FMN binding. Structural changes upon cofactor binding are compared with the monomeric flavodoxins. Topologically non-equivalent residues undergo remarkably similar local structural changes upon FMN binding to WrbA or to flavodoxin, despite differences in multimeric organization and residue types at the binding sites. Analysis of the three crystal structures described here, together with flavodoxin structures, rationalizes functional similarities and differences of the WrbAs relative to flavodoxins, leading to a new understanding of the defining features of WrbAs. The results suggest that WrbAs are not a remote and unusual branch of the flavodoxin family as previously thought but rather a central member with unifying structural features.
Asunto(s)
Cristalografía por Rayos X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Anabaena/química , Apoproteínas/química , Apoproteínas/metabolismo , Sitios de Unión , Mononucleótido de Flavina/química , Mononucleótido de Flavina/metabolismo , Flavodoxina/química , Flavodoxina/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de ProteínaRESUMEN
The crystal structure of the flavodoxin-like protein WrbA with oxidized FMN bound reveals a close relationship to mammalian NAD(P)H:quinone oxidoreductase, Nqo1. Structural comparison of WrbA, flavodoxin, and Nqo1 indicates how the twisted open-sheet fold of flavodoxins is elaborated to form multimers that extend catalytic function from one-electron transfer between protein partners using FMN to two-electron reduction of xenobiotics using FAD. The structure suggests a novel physiological role for WrbA and Nqo1.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Escherichia coli/química , Flavodoxina/química , NAD(P)H Deshidrogenasa (Quinona)/química , Proteínas Represoras/química , Sitios de Unión , Modelos Moleculares , Pliegue de ProteínaRESUMEN
The flavoprotein WrbA from Escherichia coli is considered to be the prototype of a new family of multimeric flavodoxin-like proteins that are implicated in cell protection against oxidative stress. The present study is aimed at structural characterization of the E. coli protein with respect to its recently revealed oxidoreductase activity. Crystals of WrbA holoprotein in complex with the oxidized flavin cofactor (FMN) were obtained using standard vapour-diffusion techniques. Deep yellow tetragonal crystals obtained from differing crystallization conditions display different space groups and unit-cell parameters. X-ray crystal structures of the WrbA holoprotein have been determined to resolutions of 2.0 and 2.6 A.
