title: Dynamic equilibrium between closed and partially closed states of the bacterial Enzyme I unveiled by solution NMR and X-ray scattering.
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.
authors: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520; Department of Chemistry, Iowa State University, Ames, IA 50011;
authors: Vincenzo
authors: V
authors: Venditti
authors: Division of Computational Biosciences, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892-5624.
authors: Charles D
authors: CD
authors: Schwieters
authors: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520;
authors: Alexander
authors: A
authors: Grishaev
authors: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520; mariusc@mail.nih.gov.
authors: G Marius
authors: GM
authors: Clore
mesh_terms: D000465:Algorithms; D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009584:Nitrogen / Q000737:chemistry; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry*; D010766:Phosphorylation; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000737:chemistry*; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D015398:Signal Transduction; D014965:X-Rays
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase; D009584:Nitrogen
keywords: X-ray scattering; conformational states; dipolar couplings; ligand binding; multidomain protein dynamics