biostudies-literatureVeggiani GNational Institute of General Medical SciencesCanadian Institutes of Health ResearchNIGMS NIH HHSCIHR941-956https://www.ebi.ac.uk/biostudies/studies/S-EPMC9305627biostudies-literatureUnknown17(4)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 ACS chemical biologyPanel of Engineered Ubiquitin Variants Targeting the Family of Human Ubiquitin Interacting Motifs.PMC9305627MOP-93684P30 GM124165Manczyk NKurinov ISidhu SSVeggiani GSinger AUSicheri FMartyn GDYates BPfalsePanel of Engineered Ubiquitin Variants Targeting the Family of Human Ubiquitin Interacting Motifs.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 2022-01-01T00:00:00Z2022 Apr2025-04-19T14:17:59.475Z2025-02-19T04:46:16.993ZS-EPMC93056273538564610.1021/acschembio.2c00089