Cooperative kinetics of both Hsp104 ATPase domains and interdomain communication revealed by AAA sensor-1 mutants
Cooperative kinetics of both Hsp104 ATPase domains and interdomain communication revealed by AAA sensor-1 mutants
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DOI：
10.1093/emboj/21.1.12
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发表时间：
2002-01-15
期刊：
影响因子：
11.4
通讯作者：
Lindquist, SL
中科院分区：
文献类型：
--
作者：
Hattendorf, DA;Lindquist, SL
AAA蛋白共有一个保守的ATP水解活性位点，并调节许多细胞过程。AAA蛋白是寡聚体，并且通常每个单体具有多个ATP酶结构域，这表明ATP水解的复杂变构动力学。在这里，使用野生型热休克蛋白104的六聚体状态，我们证明，它的两个AAA模块（NBD 1和NBD 2）有非常不同的催化活性，但每个显示协同水解动力学。使用突变的AAA传感器-1基序的NBD 1和NBD 2，降低ATP水解的速率，而不影响核苷酸结合，我们还研究了保持每个网站在ATP结合状态的后果。在体外，降低NBD 2的k（cat）显著改变了NBD 1的稳态动力学行为。因此，Hsp 104除了在NBD 1和NBD 2处均表现出同型协同性之外，还表现出两个位点之间的变构通信。在体内，每个传感器-1突变导致的Hsp 104功能（耐热性和酵母朊病毒繁殖）的两个试验中的功能丧失表型，证明ATP水解的重要性，不同的ATP结合在每个网站的Hsp 104功能。
AAA proteins share a conserved active site for ATP hydrolysis and regulate many cellular processes. AAA proteins are oligomeric and often have multiple ATPase domains per monomer, which is suggestive of complex allosteric kinetics of ATP hydrolysis. Here, using wild-type Hsp104 in the hexameric state, we demonstrate that its two AAA modules (NBD1 and NBD2) have very different catalytic activities, but each displays cooperative kinetics of hydrolysis. Using mutations in the AAA sensor-1 motif of NBD1 and NBD2 that reduce the rate of ATP hydrolysis without affecting nucleotide binding, we also examine the consequences of keeping each site in the ATP-bound state. In vitro, reducing k(cat) at NBD2 significantly alters the steady-state kinetic behavior of NBD1. Thus, Hsp104 exhibits allosteric communication between the two sites in addition to homotypic cooperativity at both NBD1 and NBD2. In vivo, each sensor-1 mutation causes a loss-of-function phenotype in two assays of Hsp104 function (thermotolerance and yeast prion propagation), demonstrating the importance of ATP hydrolysis as distinct from ATP binding at each site for Hsp104 function.