Pan Jonathan L, Sliskovic Inga, Bardwell James C A
Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI 48109, USA.
J Mol Biol. 2008 Apr 11;377(5):1433-42. doi: 10.1016/j.jmb.2008.01.058. Epub 2008 Jan 31.
Disulfide bond formation occurs in secreted proteins in Escherichia coli when the disulfide oxidoreductase DsbA, a soluble periplasmic protein, nonspecifically transfers a disulfide to a substrate protein. The catalytic disulfide of DsbA is regenerated by the inner-membrane protein DsbB. To help identify the specificity determinants in DsbB and to understand the nature of the kinetic barrier preventing direct oxidation of newly secreted proteins by DsbB, we imposed selective pressure to find novel mutations in DsbB that would function to bypass the need for the disulfide carrier DsbA. We found a series of mutations localized to a short horizontal alpha-helix anchored near the outer surface of the inner membrane of DsbB that eliminated the need for DsbA. These mutations changed hydrophobic residues into nonhydrophobic residues. We hypothesize that these mutations may act by decreasing the affinity of this alpha-helix to the membrane. The DsbB mutants were dependent on the disulfide oxidoreductase DsbC, a soluble periplasmic thiol-disulfide isomerase, for complementation. DsbB is not normally able to oxidize DsbC, possibly due to a steric clash that occurs between DsbC and the membrane adjacent to DsbB. DsbC must be in the reduced form to function as an isomerase. In contrast, DsbA must remain oxidized to function as an oxidizing thiol-disulfide oxidoreductase. The lack of interaction that normally exists between DsbB and DsbC appears to provide a means to separate the DsbA-DsbB oxidation pathway and the DsbC-DsbD isomerization pathway. Our mutants in DsbB may act by redirecting oxidant flow to take place through the isomerization pathway.
当二硫键氧化还原酶DsbA（一种可溶性周质蛋白）将二硫键非特异性地转移到底物蛋白上时，大肠杆菌分泌蛋白中会形成二硫键。DsbA的催化二硫键由内膜蛋白DsbB再生。为了帮助确定DsbB中的特异性决定因素，并了解阻止DsbB直接氧化新分泌蛋白的动力学障碍的性质，我们施加了选择性压力，以寻找DsbB中的新突变，这些突变将起到绕过对二硫键载体DsbA需求的作用。我们发现一系列突变定位于DsbB内膜外表面附近锚定的短水平α-螺旋上，这些突变消除了对DsbA的需求。这些突变将疏水残基变为非疏水残基。我们假设这些突变可能通过降低该α-螺旋与膜的亲和力来起作用。DsbB突变体依赖于二硫键氧化还原酶DsbC（一种可溶性周质硫醇-二硫键异构酶）进行互补。DsbB通常无法氧化DsbC，这可能是由于DsbC与DsbB相邻的膜之间发生了空间冲突。DsbC必须处于还原形式才能作为异构酶发挥作用。相比之下，DsbA必须保持氧化状态才能作为氧化硫醇-二硫键氧化还原酶发挥作用。DsbB和DsbC之间通常不存在的相互作用似乎提供了一种将DsbA-DsbB氧化途径和DsbC-DsbD异构化途径分开的方法。我们在DsbB中的突变可能通过将氧化剂流重定向通过异构化途径来起作用。
