Panel 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 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.