Panel of Engineered Ubiquitin Variants Targeting the Family ofHuman Ubiquitin Interacting Motifs

Ubiquitin (Ub)-binding domains embedded inintracellular proteins act as readers of the complex Ub code andcontribute to regulation of numerous eukaryotic processes. Ub-interacting motifs (UIMs) are short alpha-helical modular recognitionelements whose role in controlling proteostasis and signaltransduction has been poorly investigated. Moreover, impaired oraberrant activity of UIM-containing proteins has been implicated innumerous diseases, but targeting modular recognition elements inproteins remains a major challenge. To overcome this limitation, wedeveloped Ub variants (UbVs) that bind to 42 UIMs in the humanproteome with high affinity and specificity. Structural analysis of aUbV:UIM complex revealed the molecular determinants ofenhanced affinity and specificity. Furthermore, we showed that a UbV targeting a UIM in the cancer-associated Ub-specificprotease 28 potently inhibited catalytic activity. Our work demonstrates the versatility of UbVs to target short alpha-helical Ub receptorswith high affinity and specificity. Moreover, the UbVs provide a toolkit to investigate the role of UIMs in regulating and transducingUb signals and establish a general strategy for the systematic development of probes for Ub-binding domains.