Amor Alvaro J, Schmitz Karl R, Sello Jason K, Baker Tania A, Sauer Robert T
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
Department of Chemistry, Brown University, Providence, RI 02912.
ACS Chem Biol. 2016 Jun 17;11(6):1552-1560. doi: 10.1021/acschembio.6b00083. Epub 2016 Mar 30.
The ClpXP protease assembles in a reaction in which an ATP-bound ring hexamer of ClpX binds to one or both heptameric rings of the ClpP peptidase. Contacts between ClpX IGF-loops and clefts on a ClpP ring stabilize the complex. How ClpXP stability is maintained during the ATP-hydrolysis cycle that powers mechanical unfolding and translocation of protein substrates is poorly understood. Here, we use a real-time kinetic assay to monitor the effects of nucleotides on the assembly and disassembly of ClpXP. When ATP is present, complexes containing single-chain ClpX assemble via an intermediate and remain intact until transferred into buffers containing ADP or no nucleotides. ATP binding to high-affinity subunits of the ClpX hexamer prevents rapid dissociation, but additional subunits must be occupied to promote assembly. Small-molecule acyldepsipeptides, which compete with the IGF loops of ClpX for ClpP-cleft binding, cause exceptionally rapid dissociation of otherwise stable ClpXP complexes, suggesting that the IGF-loop interactions with ClpP must be highly dynamic. Our results indicate that the ClpX hexamer spends almost no time in an ATP-free state during the ATPase cycle, allowing highly processive degradation of protein substrates.
ClpXP蛋白酶在一种反应中组装，其中ATP结合的ClpX六聚体环与ClpP肽酶的一个或两个七聚体环结合。ClpX的IGF环与ClpP环上的裂缝之间的接触稳定了复合物。在为蛋白质底物的机械展开和转运提供动力的ATP水解循环过程中，ClpXP的稳定性是如何维持的，目前还知之甚少。在这里，我们使用实时动力学分析来监测核苷酸对ClpXP组装和解聚的影响。当存在ATP时，包含单链ClpX的复合物通过一个中间体组装，并保持完整，直到转移到含有ADP或不含核苷酸的缓冲液中。ATP与ClpX六聚体的高亲和力亚基结合可防止快速解离，但必须占据更多亚基才能促进组装。小分子酰基二肽与ClpX的IGF环竞争ClpP裂缝结合，导致原本稳定的ClpXP复合物异常快速解离，这表明IGF环与ClpP的相互作用一定是高度动态的。我们的结果表明，在ATP酶循环过程中，ClpX六聚体几乎没有时间处于无ATP状态，从而允许对蛋白质底物进行高度连续的降解。
