Probing the Flexibility of the DsbA Oxidoreductase from Vibrio cholerae-a 15N - 1H Heteronuclear NMR Relaxation Analysis of Oxidized and Reduced Forms of DsbA

Henry James Horne, Edward J d'Auvergne, Murray Coles, Tony Velkov, Yanni Chin, William Neil Charman, Richard John Prankerd, Paul Gooley, Martin Scanlon

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18 Citations (Scopus)

Abstract

We have determined the structure of the reduced form of the DsbA oxidoreductase from Vibrio cholerae. the reduced structure shoes a high level of similarity to the crystal structure of the oxidized form and is typical of this class of enzyme containing a thioredoxin domain with an inserted alpha-helocal domain. Proteolytic and thermal stability measurements show that the reduced form of DsbA is considerbaly more stable than the oxidized form. NMR relaxation data have been collected and analyzed using a model-free approach to probe the dynamics of the reduced and oxidized states of DsbA. Akaike s information criteria have been applied both in the selection of the model-free models and the diffusion tensors that describe the global motions of each redox form. Analysis of the dynamics reveals that the oxidized protein shows increased disorder on the oico- to nanosecond and micro- to millisecond timescale. many significant changes in dynamics are located either close to the active site or at the intersection points between the domains. In addition, analysis of the diffusion data shows there is a clear difference in the degree of interdomain movement between oxidized and reduced DsbA with the oxidized form being the more rigid. Principal components analysis has been employed to indicate possible concerted movements in the DsbA structure, which suggests that the modeled interdomain motions affect affect the catalytic cleft of the enzyme. taken together, these data provide compelling evidence of a role for dynamics in the catalytic cycle of DsbA.
Original languageEnglish
Pages (from-to)703 - 716
Number of pages14
JournalJournal of Molecular Biology
Volume371
Issue number3
Publication statusPublished - 2007

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