Dynamic equilibrium between closed and partially closed states of the bacterial Enzyme I unveiled by solution NMR and X-ray scattering.

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TitleDynamic equilibrium between closed and partially closed states of the bacterial Enzyme I unveiled by solution NMR and X-ray scattering.
Publication TypeJournal Article
Year of Publication2015
AuthorsVenditti, V, Schwieters, CD, Grishaev, A, G Clore, M
JournalProc Natl Acad Sci U S A
Volume112
Issue37
Pagination11565-70
Date Published2015 Sep 15
ISSN1091-6490
KeywordsAlgorithms, Bacterial Proteins, Catalytic Domain, Escherichia coli, Ligands, Magnetic Resonance Spectroscopy, Molecular Dynamics Simulation, Mutation, Nitrogen, Phosphoenolpyruvate Sugar Phosphotransferase System, Phosphorylation, Phosphotransferases (Nitrogenous Group Acceptor), Protein Binding, Protein Multimerization, Protein Structure, Secondary, Scattering, Radiation, Signal Transduction, X-Rays
Abstract

Enzyme I (EI) is the first component in the bacterial phosphotransferase system, a signal transduction pathway in which phosphoryl transfer through a series of bimolecular protein-protein interactions is coupled to sugar transport across the membrane. EI is a multidomain, 128-kDa homodimer that has been shown to exist in two conformational states related to one another by two large (50-90°) rigid body domain reorientations. The open conformation of apo EI allows phosphoryl transfer from His189 located in the N-terminal domain α/β (EIN(α/β)) subdomain to the downstream protein partner bound to the EIN(α) subdomain. The closed conformation, observed in a trapped phosphoryl transfer intermediate, brings the EIN(α/β) subdomain into close proximity to the C-terminal dimerization domain (EIC), thereby permitting in-line phosphoryl transfer from phosphoenolpyruvate (PEP) bound to EIC to His189. Here, we investigate the solution conformation of a complex of an active site mutant of EI (H189A) with PEP. Simulated annealing refinement driven simultaneously by solution small angle X-ray scattering and NMR residual dipolar coupling data demonstrates unambiguously that the EI(H189A)-PEP complex exists in a dynamic equilibrium between two approximately equally populated conformational states, one corresponding to the closed structure and the other to a partially closed species. The latter likely represents an intermediate in the open-to-closed transition.

DOI10.1073/pnas.1515366112
Alternate JournalProc. Natl. Acad. Sci. U.S.A.
Refereed DesignationRefereed
PubMed ID26305976
PubMed Central IDPMC4577164
Grant List / / Intramural NIH HHS / United States