Mutants in DsbB that appear to redirect oxidation through the disulfide isomerization pathway
Mutants in DsbB that appear to redirect oxidation through the disulfide isomerization pathway
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DOI：
10.1016/j.jmb.2008.01.058
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发表时间：
2008-04-11
影响因子：
5.6
通讯作者：
Bardwell, James C. A.
中科院分区：
文献类型：
--
作者：
Pan, Jonathan L.;Sliskovic, Inga;Bardwell, James C. A.
当可溶性质周蛋白二硫氧化还原酶DsbA非特异性地将二硫转移到底物蛋白时，大肠杆菌分泌蛋白中会形成二硫键。DsbA的催化二硫化物由膜内蛋白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屏障的性质，我们施加了选择性压力来寻找DsbB中的新突变，这些突变可以绕过对二硫化物载体DsbA的需求。我们发现一系列突变定位于DsbB内膜外表面附近的短水平（x-螺旋），从而消除了对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α -螺旋对膜的亲和力而起作用。DsbB突变体依赖于二硫氧化还原酶DsbC进行互补，DsbC是一种可溶性的质周硫醇-二硫异构酶。DsbB通常不能氧化DsbC，可能是由于DsbC和邻近DsbB的膜之间发生立体碰撞。DsbC必须以还原形式发挥异构酶的作用。相反，DsbA必须保持氧化状态才能发挥氧化硫醇二硫氧化还原酶的作用。DsbB和DsbC之间缺乏通常存在的相互作用似乎提供了一种分离DsbA-DsbB氧化途径和DsbC- dsbd异构化途径的方法。我们的DsbB突变体可能通过异构化途径重定向氧化剂流动来起作用。(C) 2008 Elsevier Ltd版权所有。
Disulfide bond formation occurs in secreted proteins in Escherichia coli when the disulfide oxidoreductase DsbA, a soluble periplasmic protein, non-specifically 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 (x-helix anchored near the outer surface of the inner membrane of DsbB that eliminated the need for DsbA. These mutations changed hydrophobic residues into non-hydrophobic 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. (C) 2008 Elsevier Ltd. All rights reserved.