Department of Manufacturing Pharmacy and Research Institute for Drug Development, Pusan National University, Busan 609-735, Republic of Korea.
J Struct Biol. 2013 Jul;183(1):1-10. doi: 10.1016/j.jsb.2013.05.013. Epub 2013 May 29.
The disulfide-bond isomerase DsbC plays a crucial role in the folding of bacterial proteins in the periplasmic space. DsbC has a V-shaped dimeric structure with two domains, and Cys98 in the C-terminal domain attacks inappropriate disulfide bonds in substrate proteins due to its high nucleophilic activity. In this article, we present the crystal structure of DsbC from Salmonella enterica serovar Typhimurium. We evaluated the conserved residues Asp95 and Arg125, which are located close to Cys98. The mutation of Asp95 or Arg125 abolished the disulfide isomerase activity of DsbC in an in vitro assay using a protein substrate, and the R125A mutation significantly reduced the chaperone activity for the substrate RNase I in vivo. Furthermore, a comparative analysis suggested that the conformation of Arg125 varies depending on the packing or protein-protein interactions. Based on these findings, we suggest that Asp95 and Arg125 modulate the pKa of Cys98 during catalysis.
二硫键异构酶 DsbC 在细菌蛋白在周质空间的折叠中起着至关重要的作用。DsbC 具有 V 形二聚体结构，由两个结构域组成，C 末端结构域中的 Cys98 由于其高亲核活性，攻击底物蛋白中不合适的二硫键。在本文中，我们展示了来自鼠伤寒沙门氏菌的 DsbC 的晶体结构。我们评估了靠近 Cys98 的保守残基 Asp95 和 Arg125。在使用蛋白质底物的体外实验中，Asp95 或 Arg125 的突变会使 DsbC 的二硫键异构酶活性失活，并且 R125A 突变会显著降低 RNase I 底物在体内的伴侣活性。此外，比较分析表明 Arg125 的构象取决于包装或蛋白质-蛋白质相互作用。基于这些发现，我们提出 Asp95 和 Arg125 在催化过程中调节 Cys98 的 pKa。
