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 Dim

详细信息    查看全文

• 作者：Yuki Takayama ; Charles D. Schwieters ; Alexander Grishaev ; Rodolfo Ghirlando ; G. Marius Clore
• 刊名：Journal of the American Chemical Society
• 出版年：2011
• 出版时间：January 26, 2011
• 年：2011
• 卷：133
• 期：3
• 页码：424-427
• 全文大小：296K
• 年卷期：v.133,no.3(January 26, 2011)
• ISSN：1520-5126

文摘

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^伪/尾 subdomain and an aspartate (Asp129) on the EIN^伪 subdomain results in a small (9掳) reorientation of the EIN^伪 and EIN^伪/尾 subdomains that is in turn propagated to a larger reorientation (26掳) 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.