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

Published

September 14, 2015

Author(s)

Alexander Grishaev, Vicenzo Venditti, Charles Schwieters, G. Marius Clore

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.

Citation

Proceedings of the National Academy of Sciences of the United States of America

Volume

112

Issue

37

Pub Type

Journals

Download Paper

https://doi.org/10.1073/pnas.1515366112

Local Download

Keywords

X-ray scattering, conformational states, dipolar couplings, ligand binding, multidomain protein dynamics.

Citation

Grishaev, A. , Venditti, V. , Schwieters, C. and Clore, G. (2015), 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 of the United States of America, [online], https://doi.org/10.1073/pnas.1515366112, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=919156 (Accessed December 15, 2024)