Johnson Jill L, Halas Agnieszka, Flom Gary
Department of Microbiology, Molecular Biology and Biochemistry and Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-3052, USA.
Mol Cell Biol. 2007 Jan;27(2):768-76. doi: 10.1128/MCB.01034-06. Epub 2006 Nov 13.
The ATP-dependent molecular chaperone Hsp90 and partner cochaperone proteins are required for the folding and activity of diverse cellular client proteins, including steroid hormone receptors and multiple oncogenic kinases. Hsp90 undergoes nucleotide-dependent conformational changes, but little is known about how these changes are coupled to client protein activation. In order to clarify how nucleotides affect Hsp90 interactions with cochaperone proteins, we monitored assembly of wild-type and mutant Hsp90 with Sti1, Sba1, and Cpr6 in Saccharomyces cerevisiae cell extracts. Wild-type Hsp90 bound Sti1 in a nucleotide-independent manner, while Sba1 and Cpr6 specifically and independently interacted with Hsp90 in the presence of the nonhydrolyzable analog of ATP, AMP-PNP. Alterations in Hsp90 residues that contribute to ATP binding or hydrolysis prevented or altered Sba1 and Cpr6 interaction; additional alterations affected the specificity of Cpr6 interaction. Some mutant forms of Hsp90 also displayed reduced Sti1 interaction in the presence of a nucleotide. These studies indicate that cycling of Hsp90 between the nucleotide-free, open conformation and the ATP-bound, closed conformation is influenced by residues both within and outside the N-terminal ATPase domain and that these conformational changes have dramatic effects on interaction with cochaperone proteins.
ATP 依赖性分子伴侣 Hsp90 及其伴侣共伴侣蛋白是多种细胞客户蛋白折叠和活性所必需的，这些客户蛋白包括类固醇激素受体和多种致癌激酶。Hsp90 会经历核苷酸依赖性构象变化，但对于这些变化如何与客户蛋白激活相偶联却知之甚少。为了阐明核苷酸如何影响 Hsp90 与共伴侣蛋白的相互作用，我们在酿酒酵母细胞提取物中监测了野生型和突变型 Hsp90 与 Sti1、Sba1 和 Cpr6 的组装情况。野生型 Hsp90 以核苷酸非依赖性方式结合 Sti1，而 Sba1 和 Cpr6 在 ATP 的不可水解类似物 AMP-PNP 存在的情况下特异性且独立地与 Hsp90 相互作用。对有助于 ATP 结合或水解的 Hsp90 残基进行改变会阻止或改变 Sba1 和 Cpr6 的相互作用；进一步的改变会影响 Cpr6 相互作用的特异性。一些 Hsp90 的突变形式在有核苷酸存在的情况下也表现出与 Sti1 的相互作用减少。这些研究表明，Hsp90 在无核苷酸的开放构象和 ATP 结合的封闭构象之间的循环受到 N 端 ATP 酶结构域内外残基的影响，并且这些构象变化对与共伴侣蛋白的相互作用有显著影响。
