Identification of conserved residues required for the binding of a tetratricopeptide repeat domain to heat shock protein 90
Identification of conserved residues required for the binding of a tetratricopeptide repeat domain to heat shock protein 90
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DOI：
10.1074/jbc.274.29.20060
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发表时间：
1999-07-16
影响因子：
4.8
通讯作者：
Chinkers, M
中科院分区：
文献类型：
--
作者：
Russell, LC;Whitt, SR;Chinkers, M
热休克蛋白90(HSP90)与一系列四肽重复序列(TPR)蛋白的顺序结合是其分子伴侣功能的关键。我们使用定点突变来阐明HSP90与磷酸蛋白磷酸酶5(PP5)的TPR结构域结合的结构基础。选择这个TPR结构域进行研究是因为它的三维结构是已知的。我们检测了HSP90与野生型和突变型TPR构建体从转基因细胞中的免疫共沉淀。只有位于TPR结构域一侧的突变才会影响HSP90的结合。这使得能够识别结合槽。在HSP90结合的TPR蛋白中高度保守的三个碱性残基突出地延伸到这个凹槽中。Lys-97和Arg-101是HSP90结合所必需的，而Arg-74的突变减少了，但没有取消HSP90的结合。结合槽中另一个保守的碱性残基Lys-32的突变也阻止了HSP90的结合。PP5的TPR结构域与HSP90的一个12 kDa的C末端片段特异结合。HSP90片段中酸性残基的突变降低了这种结合。这些数据表明，在与HSP90结合的TPR蛋白中，含有与HSP90 C末端附近的酸性残基相互作用的碱性残基的结合槽是保守的。
The sequential binding of heat shock protein 90 (hsp90) to a series of tetratricopeptide repeat (TPR) proteins is critical to its function as a molecular chaperone. We have used site-directed mutagenesis to clarify the structural basis for the binding of hsp90 to the TPR domain of phosphoprotein phosphatase 5 (PP5). This TPR domain was chosen for study because its three-dimensional structure is known. We examined co-immunoprecipitation of hsp90 with wild type and mutant TPR constructs from transfected cells. Only mutations located on one face of the TPR domain affected hsp90 binding. This allowed the identification of a binding groove. Three basic residues that are highly conserved in hsp90-binding TPR proteins extend prominently into this groove. Lys-97 and Arg-101 were absolutely required for hsp90 binding, while mutation of Arg-74 diminished, but did not abrogate, hsp90 binding. Mutation of Lys-32, another conserved basic residue in the binding groove, also blocked hsp90 binding. The TPR domain of PP5 bound specifically to a 12-kDa C-terminal fragment of hsp90. This binding was reduced by mutation of acidic residues in the hsp90 fragment. These data suggest conservation, among hsp90-binding TPR proteins, of a binding groove containing basic residues that interact with acidic residues near the C terminus of hsp90.