title: 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.
abstract: 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.
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, United States.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Takayama
authors.forename: Charles D
authors.initials: CD
authors.lastname: Schwieters
authors.forename: Alexander
authors.initials: A
authors.lastname: Grishaev
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Ghirlando
authors.forename: G Marius
authors.initials: GM
authors.lastname: Clore
mesh_terms: D020134:Catalytic Domain; D019281:Dimerization; D006860:Hydrogen Bonding; D008958:Models, Molecular; D015335:Molecular Probes; D009154:Mutation; D010766:Phosphorylation; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000737:chemistry*; D011487:Protein Conformation; D012542:Scattering, Radiation; D014965:X-Rays
chemical_list: D015335:Molecular Probes; D017852:Phosphotransferases (Nitrogenous Group Acceptor)