The importance of ATP binding and hydrolysis by hsp90 in formation and function of protein heterocomplexes
The importance of ATP binding and hydrolysis by hsp90 in formation and function of protein heterocomplexes
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DOI：
10.1074/jbc.274.25.17525
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发表时间：
1999-06-18
影响因子：
4.8
通讯作者：
Toft, DO
中科院分区：
文献类型：
--
作者：
Grenert, JP;Johnson, BD;Toft, DO
分子伴侣HSP90能够结合和水解三磷酸腺苷。利用与之相关的一种ATPase-DNA旋转酶B的信息，我们选择了HSP90‘S ATP结合区中的三个保守残基进行突变。其中两个突变消除了核苷酸结合，而第三个突变保留了核苷酸结合，但明显缺乏ATP水解酶。我们首先分析了这些突变如何影响热休克蛋白90的S与辅助伴侣p23和Hop，以及与疏水树脂苯基琼脂糖的结合。这些实验表明，ATP的作用，特别是增加对p23的亲和力，降低对Hop和苯基琼脂糖的亲和力，是单独由ATP结合引起的。我们还测试了HSP90突变体辅助HSP70、HSP40和Hop对变性萤火虫荧光素酶进行复性的能力。虽然热休克蛋白90能够以不依赖于核苷酸的方式参与这一过程，但对三磷酸腺苷的水解力显著增强了热休克蛋白90的S效应。最后，我们组装了孕激素受体与HSP70、HSP40、Hop、Pas和野生型或突变型HSP90的异源复合体。HSP90与受体结合既不需要ATP结合，也不需要水解，但只有野生型HSP90才能获得含有p23并能与激素结合的成熟复合体。
The chaperone hsp90 is capable of binding and hydrolyzing ATP. Using information on a related ATPase, DNA gyrase B, we selected three conserved residues in hsp90's ATP-binding domain for mutation. Two off these mutations eliminate nucleotide binding, while the third retains nucleotide binding but is apparently deficient in ATP hydrolysis. We first analyzed how these mutations affect hsp90's binding to the co-chaperones p23 and Hop, and to the hydrophobic resin, phenyl-Sepharose. These experiments showed that ATP's effects, specifically, increased affinity for p23 and decreased affinity for Hop and phenyl-Sepharose, are brought on by ATP binding alone. We also tested the ability of hsp90 mutants to assist hsp70, hsp40, and Hop in the refolding of denatured firefly luciferase. While hsp90 is capable of participating in this process in a nucleotide-independent manner, the ability to hydrolyze ATP markedly potentiates hsp90's effect. Finally, we assembled progesterone receptor heterocomplexes with hsp70, hsp40, Hop, pas, and wild type or mutant hsp90. While neither ATP binding nor hydrolysis was necessary to bind hsp90 to the receptor, mature complexes containing p23 and capable of hormone binding were only obtained with wild type hsp90.