Role of the GYVG pore motif of HslU ATPase in protein unfolding and translocation for degradation by HslV peptidase
Role of the GYVG pore motif of HslU ATPase in protein unfolding and translocation for degradation by HslV peptidase
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DOI：
10.1074/jbc.m500035200
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发表时间：
2005-06-17
影响因子：
4.8
通讯作者：
Chung, CH
中科院分区：
文献类型：
--
作者：
Park, EY;Rho, YM;Chung, CH
HslVU是由HslU ATP酶和HslV肽酶组成的ATP依赖性蛋白酶。在HslVU复合物中，HslU六聚体和HslV十二聚体的中心孔对齐，并且蛋白水解活性位点被隔离在HslV的内室中。因此，天然折叠蛋白的降解需要解折叠和易位过程才能进入HslV的蛋白水解室。高度保守的GYVG（93）序列构成HslU ATP酶的中心孔。为了确定孔基序对蛋白质解折叠和易位的作用，我们在基序中产生了各种突变，并检查了它们对HslU支持HslV对三种不同底物的蛋白水解活性的能力的影响：作为天然折叠蛋白的苏拉，作为未折叠多肽的酪蛋白和小肽。Gly残基和第91位氨基酸的芳香环结构提供的柔性是苏拉降解的必要条件。GYVG基序的相同结构特征对于酪蛋白的降解是高度优选的，尽管不是必需的。相比之下，肽水解不需要任何特征。HslU的GYVG基序的突变也显示出其ATP酶活性、对ADP的亲和力以及与HslV的相互作用的显著影响。这些结果表明HslU的GYVG基序在天然折叠蛋白的解折叠以及解折叠蛋白的易位以被HslV降解中起重要作用。这些结果还暗示了孔基序在ATP切割和HslVU复合物组装中的作用。
HslVU is an ATP-dependent protease consisting of HslU ATPase and HslV peptidase. In an HslVU complex, the central pores of HslU hexamer and HslV dodecamer are aligned and the proteolytic active sites are sequestered in the inner chamber of HslV. Thus, the degradation of natively folded proteins requires unfolding and translocation processes for their access into the proteolytic chamber of HslV. A highly conserved GYVG(93) sequence constitutes the central pore of HslU ATPase. To determine the role of the pore motif on protein unfolding and translocation, we generated various mutations in the motif and examined their effects on the ability of HslU in supporting the proteolytic activity of HslV against three different substrates: SulA as a natively folded protein, casein as an unfolded polypeptide, and a small peptide. Flexibility provided by Gly residues and aromatic ring structures of the 91st amino acid were essential for degradation of SulA. The same structural features of the GYVG motif were highly preferred, although not essential, for degradation of casein. In contrast, none of the features were required for peptide hydrolysis. Mutations in the GYVG motif of HslU also showed marked influence on its ATPase activity, affinity to ADP, and interaction with HslV. These results suggest that the GYVG motif of HslU plays important roles in unfolding of natively folded proteins as well as in translocation of unfolded proteins for degradation by HslV. These results also implicate a role of the pore motif in ATP cleavage and in the assembly of HslVU complex.