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

Proc Natl Acad Sci U S A. 2015 Sep 15;112(37):11565-70. doi: 10.1073/pnas.1515366112. Epub 2015 Aug 24.


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.

Keywords: X-ray scattering; conformational states; dipolar couplings; ligand binding; multidomain protein dynamics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Algorithms
  • Bacterial Proteins / chemistry*
  • Catalytic Domain
  • Escherichia coli / enzymology*
  • Ligands
  • Magnetic Resonance Spectroscopy
  • Molecular Dynamics Simulation
  • Mutation
  • Nitrogen / chemistry
  • Phosphoenolpyruvate Sugar Phosphotransferase System / chemistry*
  • Phosphorylation
  • Phosphotransferases (Nitrogenous Group Acceptor) / chemistry*
  • Protein Binding
  • Protein Multimerization
  • Protein Structure, Secondary
  • Scattering, Radiation
  • Signal Transduction
  • X-Rays


  • Bacterial Proteins
  • Ligands
  • Phosphoenolpyruvate Sugar Phosphotransferase System
  • Phosphotransferases (Nitrogenous Group Acceptor)
  • phosphoenolpyruvate-protein phosphotransferase
  • Nitrogen

Associated data

  • PDB/2N5T