Discovery of peptide ligands targeting a specific ubiquitin-like domain–binding site in the deubiquitinase USP11

Abstract

Ubiquitin-specific proteases (USPs) reverse ubiquitination and regulate virtually all cellular processes. Defined noncatalytic domains in USP4 and USP15 are known to interact with E3 ligases and substrate recruitment factors. No such interactions have been reported for these domains in the paralog USP11, a key regulator of DNA double-strand break repair by homologous recombination. We hypothesized that USP11 domains adjacent to its protease domain harbor unique peptide-binding sites. Here, using a next-generation phage display (NGPD) strategy, combining phage display library screening with next-generation sequencing, we discovered unique USP11-interacting peptide motifs. Isothermal titration calorimetry disclosed that the highest affinity peptides (KD of ∼10 μm) exhibit exclusive selectivity for USP11 over USP4 and USP15 in vitro. Furthermore, a crystal structure of a USP11–peptide complex revealed a previously unknown binding site in USP11's noncatalytic ubiquitin-like (UBL) region. This site interacted with a helical motif and is absent in USP4 and USP15. Reporter assays using USP11-WT versus a binding pocket–deficient double mutant disclosed that this binding site modulates USP11's function in homologous recombination–mediated DNA repair. The highest affinity USP11 peptide binder fused to a cellular delivery sequence induced significant nuclear localization and cell cycle arrest in S phase, affecting the viability of different mammalian cell lines. The USP11 peptide ligands and the paralog-specific functional site in USP11 identified here provide a framework for the development of new biochemical tools and therapeutic agents. We propose that an NGPD-based strategy for identifying interacting peptides may be applied also to other cellular targets.

Small molecules that target the ubiquitin system

“…One way to obtain selective inhibitors would be to target the non-catalytic regions rather than catalytic pockets. For instance, USP7 contains a central binding cleft in the N-terminal TRAF-like domain that is specifically recognized by [PA]-xx-S amino acid motifs found in p53, Hdm2, and Epstein-Barr nuclear antigen 1 [91]; the rhodanese domain of USP8 includes a ligase recognition site [124]; a non-catalytic ubiquitin-like (UBL) region of USP11 has been shown to interact with a helical motif but this site is absent from paralogs USP4 and USP15 [125]. However, current knowledge of such binding sites is still lacking for most DUBs and E3 ligases.…”

DUBbing Down Translation: The Functional Interaction of Deubiquitinases with the Translational Machinery

“…Using a ubiquitin chain cleavage assay, Harper and colleagues reported that USP11 may cleave a panel of Lys (48, 63, 6, 33, and others)-linked linear ubiquitin chains, indicating that USP11 may function in regulating protein activity (51). Recently, Dreveny and her group reported the identification of peptide ligands that exclusively targeted the UBL domain of USP11 without affecting its functional paralogs USP4 and USP15, providing an ideal platform to next-generation biochemical tools for limiting USP11 activity (52). Thus, there are exciting opportunities to specifically target USP11 with minimal off-target impact.…”

Putting phage to work in deubiquitinase ligand discovery

“…Spiliotopoulos et al (5) next analyzed two prototype peptides for USP11 binding and function. In vitro, the peptides bound USP11 with an equilibrium dissociation constant (K D ) of ϳ10 M, and were specific, unable to bind the USP4 and USP15 paralogs.…”