The bacterial phosphotransferase system couples phosphoryl transfer to sugar transport across the cell membrane. The first protein in the pathway, Enzyme I (EI), undergoes two large rigid body domain reorientations between an autophosphorylation-competent closed state and an open state that allows subsequent phosphoryl transfer to its downstream protein partner. Simultaneous use of solution X-ray scattering and NMR dipolar coupling data to guide simulated annealing refinement reveals the existence of a dynamic equilibrium between closed and partially closed conformations in a complex of a mutant of EI with phosphoenolpyruvate. The partially closed conformation represents an intermediate in the open-to-closed transition

Clore, G.

Grishaev, Alexander

Schwieters, Charles

Venditti, Vincenzo

English

PubMed

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 (EINα/β) subdomain to the downstream protein partner bound to the EINα subdomain. The closed conformation, observed in a trapped phosphoryl transfer intermediate, brings the EINα/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 transitionThis is an article from Venditti, Vincenzo, Charles D. Schwieters, Alexander Grishaev, and G. Marius Clore. "Dynamic equilibrium between closed and partially closed states of the bacterial Enzyme I unveiled by solution NMR and X-ray scattering." Proceedings of the National Academy of Sciences 112, no. 37 (2015): 11565-11570. doi: 10.1073/pnas.1515366112. Posted with permission.</p

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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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          The bacterial phosphotransferase system couples phosphoryl transfer to sugar transport across the cell membrane. The first protein in the pathway, Enzyme I (EI), undergoes two large rigid body domain reorientations between an autophosphorylation-competent closed state and an open state that allows subsequent phosphoryl transfer to its downstream protein partner. Simultaneous use of solution X-ray scattering and NMR dipolar coupling data to guide simulated annealing refinement reveals the existence of a dynamic equilibrium between closed and partially closed conformations in a complex of a mutant of EI with phosphoenolpyruvate. The partially closed conformation represents an intermediate in the open-to-closed transition.</jats:p

Vincenzo Venditti

Charles D. Schwieters

Alexander Grishaev

G. Marius Clore

Crossref

Proceedings of the National Academy of Sciences

https://dr.lib.iastate.edu/handle/20.500.12876/14294

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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Digital Repository @ Iowa State University (ISU)

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