More than half of the ~20,000 protein-encoding human genes have paralogs. Chemical proteomics has uncovered many electrophile-sensitive cysteines that are exclusive to subsets of paralogous proteins. Here we explore whether such covalent compound–cysteine interactions can be used to discover ligandable pockets in paralogs lacking the cysteine. Leveraging the covalent ligandability of C109 in the cyclin CCNE2, we substituted the corresponding residue in paralog CCNE1 to cysteine (N112C) and found through activity-based protein profiling that this mutant reacts stereoselectively and site-specifically with tryptoline acrylamides. We then converted the tryptoline acrylamide–CCNE1-N112C interaction into in vitro NanoBRET (bioluminescence resonance energy transfer) and in cellulo activity-based protein profiling assays capable of identifying compounds that reversibly inhibit both the N112C mutant and wild-type CCNE1:CDK2 (cyclin-dependent kinase 2) complexes. X-ray crystallography revealed a cryptic allosteric pocket at the CCNE1:CDK2 interface adjacent to N112 that binds the reversible inhibitors. Our findings, thus, show how electrophile–cysteine interactions mapped by chemical proteomics can extend the understanding of protein ligandability beyond covalent chemistry.

大约 20,000 个编码蛋白质的人类基因中，超过一半具有旁系同源基因。化学蛋白质组学发现了许多亲电子敏感的半胱氨酸，这些半胱氨酸是旁系同源蛋白质子集所独有的。在这里，我们探讨这种共价化合物-半胱氨酸相互作用是否可用于发现缺乏半胱氨酸的旁系同源物中的可配位口袋。利用细胞周期蛋白 CCNE2 中 C109 的共价配位性，我们将旁系同源物 CCNE1 中的相应残基替换为半胱氨酸 (N112C)，并通过基于活性的蛋白质分析发现该突变体与色氨酸丙烯酰胺发生立体选择性和位点特异性反应。然后，我们将色氨酸丙烯酰胺-CCNE1-N112C 相互作用转化为体外 NanoBRET（生物发光共振能量转移）和基于纤维素活性的蛋白质分析测定，能够识别可逆抑制 N112C 突变体和野生型 CCNE1:CDK2（细胞周期蛋白）的化合物。 -依赖性激酶2)复合物。 X 射线晶体学揭示了 CCNE1:CDK2 界面上与 N112 相邻的神秘变构袋，该变构袋与可逆抑制剂结合。因此，我们的研究结果表明，化学蛋白质组学绘制的亲电子-半胱氨酸相互作用如何能够将蛋白质配体性的理解扩展到共价化学之外。