title: Saturation scanning of ubiquitin variants reveals a common hot spot for binding to USP2 and USP21.
abstract: A detailed understanding of the molecular mechanisms whereby ubiquitin (Ub) recognizes enzymes in the Ub proteasome system is crucial for understanding the biological function of Ub. Many structures of Ub complexes have been solved and, in most cases, reveal a large structural epitope on a common face of the Ub molecule. However, owing to the generally weak nature of these interactions, it has been difficult to map in detail the functional contributions of individual Ub side chains to affinity and specificity. Here we took advantage of Ub variants (Ubvs) that bind tightly to particular Ub-specific proteases (USPs) and used phage display and saturation scanning mutagenesis to comprehensively map functional epitopes within the structural epitopes. We found that Ubvs that bind to USP2 or USP21 contain a remarkably similar core functional epitope, or "hot spot," consisting mainly of positions that are conserved as the wild type sequence, but also some positions that prefer mutant sequences. The Ubv core functional epitope contacts residues that are conserved in the human USP family, and thus it is likely important for the interactions of Ub across many family members.
authors.affiliation: Banting and Best Department of Medical Research and Department of Molecular Genetics, The Donnelly Centre, University of Toronto, Toronto, ON, Canada M5S 3E1;
authors.forename: Isabel
authors.initials: I
authors.lastname: Leung
authors.affiliation: Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
authors.forename: Ayelet
authors.initials: A
authors.lastname: Dekel
authors.affiliation: Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 9190401, Israel jshifman@mail.huji.ac.il sachdev.sidhu@utoronto.ca.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Shifman
authors.affiliation: Banting and Best Department of Medical Research and Department of Molecular Genetics, The Donnelly Centre, University of Toronto, Toronto, ON, Canada M5S 3E1; jshifman@mail.huji.ac.il sachdev.sidhu@utoronto.ca.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003198:Computer Simulation; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D000072417:Protein Domains; D017386:Sequence Homology, Amino Acid; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D000939:Epitopes; C408875:USP21 protein, human; D025801:Ubiquitin; D010450:Endopeptidases; C108665:USP2 protein, human; D043222:Ubiquitin Thiolesterase
keywords: USP; hot spots; phage display; saturation scanning; ubiquitin