Allosteric Communication between the Nucleotide Binding Domains of Caseinolytic Peptidase B
Allosteric Communication between the Nucleotide Binding Domains of Caseinolytic Peptidase B
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DOI：
10.1074/jbc.m111.231365
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发表时间：
2011-07-22
影响因子：
4.8
通讯作者：
Muga, Arturo
中科院分区：
文献类型：
--
作者：
Angel Fernandez-Higuero, Jose;Acebron, Sergio P.;Muga, Arturo
ClpB是一种六聚体分子伴侣，其与Hsp 70/DnaK分子伴侣系统合作溶解并再活化蛋白质聚集体。每个相同的蛋白质单体含有两个核苷酸结合结构域（NBD），其ATP酶活性必须偶联以在底物上施加其再活化所需的机械功。然而，目前人们对这些网站之间的通信是如何发生的知之甚少。我们在本文中研究了每种NBD对WT ClpB和蛋白变体中的核苷酸的亲和力，其中一个或两个位点突变以选择性地损害核苷酸结合或水解。我们的数据表明，NBD 2对核苷酸的亲和力（Kd = 3-7 μ M）明显高于NBD 1。有趣的是，NBD 1的亲和力取决于核苷酸与NBD 2的结合。ATP而不是ADP与NBD 2的结合增加了NBD 1对相应核苷酸的亲和力（Kd从大约160-300 μ M降低到50-60 μ M）。此外，用ATP填充NBD 2环允许该核苷酸和底物与NBD 1环的协同结合。数据还表明，至少有四个亚基合作，结合和重新激活两种不同的聚集蛋白质底物。
ClpB is a hexameric chaperone that solubilizes and reactivates protein aggregates in cooperation with the Hsp70/DnaK chaperone system. Each of the identical protein monomers contains two nucleotide binding domains (NBD), whose ATPase activity must be coupled to exert on the substrate the mechanical work required for its reactivation. However, how communication between these sites occurs is at present poorly understood. We have studied herein the affinity of each of the NBDs for nucleotides in WT ClpB and protein variants in which one or both sites are mutated to selectively impair nucleotide binding or hydrolysis. Our data show that the affinity of NBD2 for nucleotides (K-d = 3-7 mu M) is significantly higher than that of NBD1. Interestingly, the affinity of NBD1 depends on nucleotide binding to NBD2. Binding of ATP, but not ADP, to NBD2 increases the affinity of NBD1 (the Kd decreases from approximate to 160-300 to 50-60 mu M) for the corresponding nucleotide. Moreover, filling of the NBD2 ring with ATP allows the cooperative binding of this nucleotide and substrates to the NBD1 ring. Data also suggest that a minimum of four subunits cooperate to bind and reactivate two different aggregated protein substrates.