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(?/?) subdomain and an aspartate (Asp129) on the EIN(?) subdomain results in a small (?9°) reorientation of the EIN(?) and EIN(?/?) 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.

SUBMITTER: Takayama Y 

PROVIDER: S-EPMC3071875 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature