Coupling ATP utilization to protein remodeling by ClpB, a hexameric AAA plus protein
Coupling ATP utilization to protein remodeling by ClpB, a hexameric AAA plus protein
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DOI：
10.1073/pnas.0911937106
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发表时间：
2009-12-29
影响因子：
11.1
通讯作者：
Wickner, Sue
中科院分区：
文献类型：
--
作者：
Hoskins, Joel R.;Doyle, Shannon M.;Wickner, Sue
ClpB 和 Hsp104 是 AAA+（与各种细胞活动相关的 ATP 酶）蛋白质家族的成员，并且是参与耐热性的分子机器。它们是六聚体蛋白质，含有 12 个 ATP 结合位点，每个原聚体有两个位点。 ClpB 和 Hsp104 具有一些先天的蛋白质重塑活性；然而，它们需要 DnaK/Hsp70 伴侣系统的协作来解聚和重新激活不溶性聚集蛋白。我们研究了 ClpB 在有或没有 DnaK 系统的情况下将 ATP 利用与蛋白质重塑耦合的机制。当野生型 ClpB（在 ATP 存在下无法单独重塑蛋白质）与无法水解 ATP 的 ClpB 突变体混合时，异六聚体令人惊讶地获得了蛋白质重塑活性。在没有 DnaK 系统的情况下，异六聚体的最佳蛋白质重塑需要大约三个活性原聚体和三个非活性原聚体。此外，六聚体中活性和非活性 ATP 结合位点的位置并不重要。结果表明，在 DnaK 系统缺失的情况下，ClpB 通过概率机制发挥作用。然而，当我们测量 ClpB 异六聚体与 DnaK 系统结合的蛋白质解聚时，单个无活性 ClpB 亚基的掺入会阻断活性，支持 ATP 利用的顺序机制。综上所述，结果表明 ClpB 利用 ATP 的机制具有适应性，并且可以根据特定底物和 DnaK 系统的存在而变化。
ClpB and Hsp104 are members of the AAA+ (ATPases associated with various cellular activities) family of proteins and are molecular machines involved in thermotolerance. They are hexameric proteins containing 12 ATP binding sites with two sites per protomer. ClpB and Hsp104 possess some innate protein remodeling activities; however, they require the collaboration of the DnaK/Hsp70 chaperone system to disaggregate and reactivate insoluble aggregated proteins. We investigated the mechanism by which ClpB couples ATP utilization to protein remodeling with and without the DnaK system. When wild-type ClpB, which is unable to remodel proteins alone in the presence of ATP, was mixed with a ClpB mutant that is unable to hydrolyze ATP, the heterohexamers surprisingly gained protein remodeling activity. Optimal protein remodeling by the heterohexamers in the absence of the DnaK system required approximately three active and three inactive protomers. In addition, the location of the active and inactive ATP binding sites in the hexamer was not important. The results suggest that in the absence of the DnaK system, ClpB acts by a probabilistic mechanism. However, when we measured protein disaggregation by ClpB heterohexamers in conjunction with the DnaK system, incorporation of a single inactive ClpB subunit blocked activity, supporting a sequential mechanism of ATP utilization. Taken together, the results suggest that the mechanism of ATP utilization by ClpB is adaptable and can vary depending on the specific substrate and the presence of the DnaK system.