Although Hsp90‐family chaperones have been extensively targeted with ATP‐competitive inhibitors, it is unknown whether high affinity is achieved from a few highly stabilizing contacts or from many weaker contacts within the ATP‐binding pocket. A large‐scale analysis of Hsp90α:inhibitor structures shows that inhibitor hydrogen‐bonding to a conserved aspartate (D93 in Hsp90α) stands out as most universal among Hsp90 inhibitors. Here we show that the D93 region makes a dominant energetic contribution to inhibitor binding for both cytosolic and organelle‐specific Hsp90 paralogs. For inhibitors in the resorcinol family, the D93:inhibitor hydrogen‐bond is pH‐dependent because the associated inhibitor hydroxyl group is titratable, rationalizing a linked‐protonation event previously observed by the Matulis group. The inhibitor hydroxyl group pK_a associated with the D93 hydrogen‐bond is therefore critical for optimizing the affinity of resorcinol derivatives, and we demonstrate that spectrophotometric measurements can determine this pK_a value. Quantifying the energetic contribution of the D93 hotspot is best achieved with the mitochondrial Hsp90 paralog, yielding 3–6 kcal/mol of stabilization (35–60% of the total binding energy) for a diverse set of inhibitors. The Hsp90 Asp93➔Asn substitution has long been known to abolish nucleotide binding, yet puzzlingly, native sequences of structurally similar ATPases, such as Topoisomerasese II, have an asparagine at this same crucial site. While aspartate and asparagine sidechains can both act as hydrogen bond acceptors, we show that a steric clash prevents the Hsp90 Asp93➔Asn sidechain from adopting the necessary rotamer, whereas this steric restriction is absent in Topoisomerasese II.

中文翻译：

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Hsp90 分子伴侣具有结合抑制剂的能量热点。

尽管 Hsp90 家族伴侣已被 ATP 竞争性抑制剂广泛靶向，但尚不清楚高亲和力是通过 ATP 结合口袋内的一些高度稳定的接触还是通过许多较弱的接触来实现的。对 Hsp90α：抑制剂结构的大规模分析表明，与保守天冬氨酸（Hsp90α 中的 D93）形成氢键的抑制剂是 Hsp90 抑制剂中最普遍的。在这里，我们表明 D93 区域对细胞质和细胞器特异性 Hsp90 旁系同源物的抑制剂结合具有显着的能量贡献。对于间苯二酚家族的抑制剂，D93：抑制剂氢键是 pH 依赖性的，因为相关抑制剂羟基是可滴定的，这合理化了 Matulis 小组先前观察到的连接质子化事件。因此，与 D93 氢键相关的抑制剂羟基pK _a对于优化间苯二酚衍生物的亲和力至关重要，我们证明分光光度测量可以确定该pK _a值。量化 D93 热点的能量贡献最好通过线粒体 Hsp90 旁系同源物来实现，为多种抑制剂产生 3-6 kcal/mol 的稳定性（总结合能的 35-60%）。人们早已知道 Hsp90 Asp93➔Asn 取代可以消除核苷酸结合，但令人困惑的是，结构相似的 ATP 酶（例如拓扑异构酶 II）的天然序列在同一关键位点上有一个天冬酰胺。虽然天冬氨酸和天冬酰胺侧链都可以充当氢键受体，但我们发现空间冲突阻止了 Hsp90 Asp93➔Asn 侧链采用必要的旋转异构体，而拓扑异构酶 II 中不存在这种空间限制。