Combined use of residual dipolar couplings and solution X-ray scattering to rapidly probe rigid-body conformational transitions in a non-phosphorylatable active-site mutant of the 128 kDa enzyme I dimer

J Am Chem Soc. 2011 Jan 26;133(3):424-7. doi: 10.1021/ja109866w. Epub 2010 Dec 16.


The first component of the bacterial phosphotransferase system, enzyme I (EI), is a multidomain 128 kDa dimer that undergoes large rigid-body conformational transitions during the course of its catalytic cycle. Here we investigate the solution structure of a non-phosphorylatable active-site mutant in which the active-site histidine is substituted by glutamine. We show that perturbations in the relative orientations and positions of the domains and subdomains can be rapidly and reliably determined by conjoined rigid-body/torsion angle/Cartesian simulated annealing calculations driven by orientational restraints from residual dipolar couplings and shape and translation information afforded by small- and wide-angle X-ray scattering. Although histidine and glutamine are isosteric, the conformational space available to a Gln side chain is larger than that for the imidazole ring of His. An additional hydrogen bond between the side chain of Gln189 located on the EIN(α/β) subdomain and an aspartate (Asp129) on the EIN(α) subdomain results in a small (∼9°) reorientation of the EIN(α) and EIN(α/β) subdomains that is in turn propagated to a larger reorientation (∼26°) of the EIN domain relative to the EIC dimerization domain, illustrating the positional sensitivity of the EIN domain and its constituent subdomains to small structural perturbations.

Publication types

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

MeSH terms

  • Catalytic Domain
  • Dimerization
  • Hydrogen Bonding
  • Models, Molecular
  • Molecular Probes
  • Mutation*
  • Phosphorylation
  • Phosphotransferases (Nitrogenous Group Acceptor) / chemistry*
  • Protein Conformation
  • Scattering, Radiation
  • X-Rays


  • Molecular Probes
  • Phosphotransferases (Nitrogenous Group Acceptor)