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.
Highlights
Ubiquitin-specific proteases (USPs) reverse ubiquitination and regulate virtually all cellular processes
We subsequently focused on the FYLIR peptide and showed that full-length ubiquitin-specific protease 11 (USP11) and its N-terminal domains alone display comparable dissociation constants indicating that the presence of the protease domain has no influence on the interaction (Fig. S2)
This study describes the discovery for the first time of unique peptide ligands for a ubiquitin-specific protease by the means of next-generation phage display
Summary
We subsequently focused on the FYLIR peptide and showed that full-length USP11 and its N-terminal domains alone display comparable dissociation constants indicating that the presence of the protease domain has no influence on the interaction (Fig. S2). Hydrogen bonding interactions between main chain carbonyls and amine groups of peptide residues Lys-9, Arg-11, and USP11 Leu-208 are key features of the interaction (Fig. 2, C and D). An interaction between FYLIR peptide and the DU domains of either USP4 or USP15 by ITC was not observed, suggesting that this binding cleft for the peptide is specific to USP11 (Fig. 4C). The arginine residues are engaged in -stacking and ion– interactions with the aromatic side chains of USP15 Phe-188 and USP4 Tyr-192 and occlude the pocket. The smaller side chains of Leu-208 and Ser-242 may be the key residues that allow USP11 to engage with the peptide ligand. To further verify the key role of this pocket for ligand binding in solution, we generated a USP11_DU L208F and S242R double mutant (USP11_DUL208F/S242R) to render this region
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