Department of Bioengineering, University of Washington, Seattle, Washington, USA.
Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
Protein Sci. 2023 Jul;32(7):e4656. doi: 10.1002/pro.4656.
Hsp90 is a molecular chaperone involved in the refolding and activation of numerous protein substrates referred to as clients. While the molecular determinants of Hsp90 client specificity are poorly understood and limited to a handful of client proteins, strong clients are thought to be destabilized and conformationally extended. Here, we measured the phosphotransferase activity of 3929 variants of the tyrosine kinase Src in both the presence and absence of an Hsp90 inhibitor. We identified 84 previously unknown functionally dependent client variants. Unexpectedly, many destabilized or extended variants were not functionally dependent on Hsp90. Instead, functionally dependent client variants were clustered in the αF pocket and β1-β2 strand regions of Src, which have yet to be described in driving Hsp90 dependence. Hsp90 dependence was also strongly correlated with kinase activity. We found that a combination of activation, global extension, and general conformational flexibility, primarily induced by variants at the αF pocket and β1-β2 strands, was necessary to render Src functionally dependent on Hsp90. Moreover, the degree of activation and flexibility required to transform Src into a functionally dependent client varied with variant location, suggesting that a combination of regulatory domain disengagement and catalytic domain flexibility are required for chaperone dependence. Thus, by studying the chaperone dependence of a massive number of variants, we highlight factors driving Hsp90 client specificity and propose a model of chaperone-kinase interactions.
Hsp90 是一种分子伴侣，参与许多被称为客户的蛋白质底物的重折叠和激活。虽然 Hsp90 客户特异性的分子决定因素了解甚少，并且仅限于少数客户蛋白，但强客户蛋白被认为是不稳定的和构象延伸的。在这里，我们在存在和不存在 Hsp90 抑制剂的情况下测量了酪氨酸激酶 Src 的 3929 种变体的磷酸转移酶活性。我们鉴定了 84 种以前未知的功能相关客户变体。出乎意料的是，许多不稳定或延伸的变体并不依赖于 Hsp90 发挥功能。相反，功能相关的客户变体聚集在 Src 的 αF 口袋和 β1-β2 链区，这些区域尚未在驱动 Hsp90 依赖性方面进行描述。Hsp90 依赖性也与激酶活性强烈相关。我们发现，激活、全局延伸和普遍构象灵活性的组合，主要是由 αF 口袋和 β1-β2 链区的变体诱导的，是使 Src 依赖于 Hsp90 发挥功能所必需的。此外，将 Src 转化为功能相关客户所需的激活和灵活性程度因变体位置而异，这表明需要调节域脱离和催化域灵活性的组合才能依赖伴侣。因此，通过研究大量变体的伴侣依赖性，我们强调了驱动 Hsp90 客户特异性的因素，并提出了伴侣-激酶相互作用的模型。
