Wang Qing, Song Changcheng, Irizarry Lauren, Dai Renming, Zhang Xiaodong, Li Chou-Chi H
Laboratory of Cancer Prevention, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland 21702, USA.
J Biol Chem. 2005 Dec 9;280(49):40515-23. doi: 10.1074/jbc.M509636200. Epub 2005 Oct 10.
The 97-kDa molecular chaperone valosin-containing protein (VCP) belongs to a highly conserved AAA family and forms a hexameric structure that is essential for its biological functions. The AAA domain contains highly conserved motifs, the Walker A, Walker B, and the second region of homology (SRH). Although Walker A and B motifs mediate ATP binding and hydrolysis, respectively, the function of the SRH in VCP is not clear. We examined the significance of the SRH in VCP, especially the conserved Arg(359) and Arg(362) in the first AAA domain, D1 and Arg(635) and Arg(638) in the second AAA domain, D2. We show that Arg(359) and Arg(362) in D1 are critical for maintaining the hexameric structure and the ability to bind the polyubiquitin chains. Although the rest of the tested SRH mutants retain the hexameric structure, all of them exhibit severely reduced ATPase activity. Tryptophan fluorescence analysis showed that all of the tested mutants can bind to ATP or ADP. Thus, the reduced ATPase activity likely results from the hampered communications among protomers during hydrolysis. Moreover, when the ATPase-defective mutant R635A or R638A is mixed with the Walker A mutant of D2, the ATPase activity is partially restored, suggesting that Arg(635) and Arg(638) can stimulate the ATPase activity of the neighboring protomer. Interestingly, mutation of Arg(359) and Arg(362) uncouples the inhibitory effect of p47, a VCP co-factor, on the ATPase activity of VCP. Therefore, the Arg residues allow D1 to take on a specific conformation that is required for substrate binding and co-factor communications. Taken together, these results demonstrate that the conserved Arg residues in the SRH of both D1 and D2 play critical roles in communicating the conformational changes required for ATP hydrolysis, and SRH in D1 also contributes to substrate binding and co-factor communications.
97千道尔顿的分子伴侣含缬酪肽蛋白（VCP）属于高度保守的AAA家族，形成一种六聚体结构，这对其生物学功能至关重要。AAA结构域包含高度保守的基序，即沃克A基序、沃克B基序和第二同源区域（SRH）。虽然沃克A基序和沃克B基序分别介导ATP结合和水解，但VCP中SRH的功能尚不清楚。我们研究了VCP中SRH的重要性，特别是第一个AAA结构域D1中保守的精氨酸（359）和精氨酸（362），以及第二个AAA结构域D2中精氨酸（635）和精氨酸（638）。我们发现，D1中的精氨酸（359）和精氨酸（362）对于维持六聚体结构以及结合多聚泛素链的能力至关重要。虽然其余测试的SRH突变体保留了六聚体结构，但它们均表现出ATP酶活性严重降低。色氨酸荧光分析表明，所有测试的突变体都能与ATP或ADP结合。因此，ATP酶活性降低可能是由于水解过程中单体之间的通讯受阻所致。此外，当ATP酶缺陷型突变体R635A或R638A与D2的沃克A突变体混合时，ATP酶活性部分恢复，这表明精氨酸（635）和精氨酸（638）可以刺激相邻单体的ATP酶活性。有趣的是，精氨酸（359）和精氨酸（362）的突变消除了VCP辅因子p47对VCP的ATP酶活性的抑制作用。因此，这些精氨酸残基使D1呈现出底物结合和辅因子通讯所需的特定构象。综上所述，这些结果表明，D1和D2的SRH中保守的精氨酸残基在传递ATP水解所需的构象变化方面起关键作用，并且D1中的SRH也有助于底物结合和辅因子通讯。
