Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, 160 College Street, Toronto, Ontario M5S3E1, Canada.
Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
ACS Chem Biol. 2022 Apr 15;17(4):941-956. doi: 10.1021/acschembio.2c00089. Epub 2022 Apr 6.
Ubiquitin (Ub)-binding domains embedded in intracellular proteins act as readers of the complex Ub code and contribute to regulation of numerous eukaryotic processes. Ub-interacting motifs (UIMs) are short α-helical modular recognition elements whose role in controlling proteostasis and signal transduction has been poorly investigated. Moreover, impaired or aberrant activity of UIM-containing proteins has been implicated in numerous diseases, but targeting modular recognition elements in proteins remains a major challenge. To overcome this limitation, we developed Ub variants (UbVs) that bind to 42 UIMs in the human proteome with high affinity and specificity. Structural analysis of a UbV:UIM complex revealed the molecular determinants of enhanced affinity and specificity. Furthermore, we showed that a UbV targeting a UIM in the cancer-associated Ub-specific protease 28 potently inhibited catalytic activity. Our work demonstrates the versatility of UbVs to target short α-helical Ub receptors with high affinity and specificity. Moreover, the UbVs provide a toolkit to investigate the role of UIMs in regulating and transducing Ub signals and establish a general strategy for the systematic development of probes for Ub-binding domains.
细胞内蛋白质中嵌入的泛素（Ub）结合域充当复杂 Ub 密码的读取器，并有助于调节众多真核生物过程。Ub 相互作用基序（UIMs）是短的 α-螺旋模块化识别元件，其在控制蛋白质稳态和信号转导中的作用尚未得到充分研究。此外，含 UIM 蛋白的活性受损或异常与许多疾病有关，但靶向蛋白质中的模块化识别元件仍然是一个主要挑战。为了克服这一限制，我们开发了 Ub 变体（UbVs），它们能够以高亲和力和特异性结合人类蛋白质组中的 42 个 UIM。对 UbV:UIM 复合物的结构分析揭示了增强亲和力和特异性的分子决定因素。此外，我们表明，靶向与癌症相关的 Ub 特异性蛋白酶 28 中的 UIM 的 UbV 可有效抑制催化活性。我们的工作证明了 UbVs 具有靶向高亲和力和特异性的短 α-螺旋 Ub 受体的多功能性。此外，UbVs 提供了一个工具包，用于研究 UIM 在调节和转导 Ub 信号中的作用，并为 Ub 结合域的系统开发探针建立了一般策略。
