Ligand Conformational Bias Drives Enantioselective Modification of a Surface-Exposed Lysine on Hsp90
Ligand Conformational Bias Drives Enantioselective Modification of a Surface-Exposed Lysine on Hsp90
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DOI：
10.1021/jacs.9b09684
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发表时间：
2020-02-19
影响因子：
15
通讯作者：
Taunton, Jack
中科院分区：
文献类型：
--
作者：
Cuesta, Adolfo;Wan, Xiaobo;Taunton, Jack
表面暴露的赖氨酸的靶向共价修饰具有挑战性，因为它们的内在反应性较低，并且在整个蛋白质组中的流行率很高。通过可逆结合的抑制剂(k(Inact))优化共价键形成速率的策略通常涉及增加电泳体的反应性，这增加了非靶标修饰的风险。在这里，我们采用了另一种方法来增加赖氨酸靶向共价Hsp90抑制剂的k(Inact)，而与可逆结合亲和力(K-I)或固有亲电性无关。从一个非共价配体开始，我们添加了一个手性的、构象受限的连接体，它使芳基磺酰氟与Hsp90表面的LysS8发生快速和对映选择性的反应。生化实验和共价和非共价配体/Hsp90络合物的高分辨晶体结构为配体构象在观察到的对映选择性中所起的作用提供了机械性的见解。最后，我们展示了细胞Hsp90的选择性共价靶向，这导致了延长的热休克反应，尽管伴随着共价配体/Hsp90复合体的降解。我们的工作突出了工程配基构象约束的潜力，以显著加速蛋白质靶标表面远端亲核性较差的赖氨酸的共价修饰。
Targeted covalent modification of surface-exposed lysines is challenging due to their low intrinsic reactivity and high prevalence throughout the proteome. Strategies for optimizing the rate of covalent bond formation by a reversibly bound inhibitor (k(inact)) typically involve increasing the reactivity of the electrophile, which increases the risk of off-target modification. Here, we employ an alternative approach for increasing k(inact) of a lysine targeted covalent Hsp90 inhibitor, independent of the reversible binding affinity (K-i) or the, intrinsic electrophilicity. Starting with a noncovalent ligand, we appended a chiral, conformationally constrained linker, which orients an arylsulfonyl fluoride to react rapidly and enantioselectively with LysS8 on the surface of Hsp90. Biochemical experiments and high-resolution crystal structures of covalent and noncovalent ligand/Hsp90 complexes provide mechanistic insights into the role of ligand conformation in the observed enantioselectivity. Finally, we demonstrate selective covalent targeting of cellular Hsp90, which results in a prolonged heat shock response despite concomitant degradation of the covalent ligand/Hsp90 complex. Our work highlights the potential of engineering ligand conformational constraints to dramatically accelerate covalent modification of a distal, poorly nucleophilic lysine on the surface of a protein target.