title: Conformational stabilization of ubiquitin yields potent and selective inhibitors of USP7.
abstract: Protein conformation and function are often inextricably linked, such that the states a protein adopts define its enzymatic activity or its affinity for various partners. Here we combine computational design with macromolecular display to isolate functional conformations of ubiquitin that tightly bind the catalytic core of the oncogenic ubiquitin-specific protease 7 (USP7) deubiquitinase. Structural and biochemical characterization of these ubiquitin variants suggest that remodeled backbone conformations and core packing poise these molecules for stronger interactions, leading to potent and specific inhibition of enzymatic activity. A ubiquitin variant expressed in human tumor cell lines binds and inhibits endogenous USP7, thereby enhancing Mdm2 proteasomal turnover and stabilizing p53. In sum, we have developed an approach to rationally target macromolecular libraries toward the remodeling of protein conformation, shown that engineering of ubiquitin conformation can greatly increase its interaction with deubiquitinases and developed powerful tools to probe the cellular role of USP7.
authors.affiliation: Department of Early Discovery Biochemistry, Genentech Inc., San Francisco, California, USA.
authors.forename: Yingnan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Lijuan
authors.initials: L
authors.lastname: Zhou
authors.forename: Lionel
authors.initials: L
authors.lastname: Rouge
authors.forename: Aaron H
authors.initials: AH
authors.lastname: Phillips
authors.forename: Cynthia
authors.initials: C
authors.lastname: Lam
authors.forename: Peter
authors.initials: P
authors.lastname: Liu
authors.forename: Wendy
authors.initials: W
authors.lastname: Sandoval
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Helgason
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Murray
authors.forename: Ingrid E
authors.initials: IE
authors.lastname: Wertz
authors.forename: Jacob E
authors.initials: JE
authors.lastname: Corn
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011480:Protease Inhibitors / Q000494:pharmacology*; D011487:Protein Conformation; D025801:Ubiquitin / Q000737:chemistry*; D043222:Ubiquitin Thiolesterase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D000076084:Ubiquitin-Specific Peptidase 7
chemical_list: D011480:Protease Inhibitors; D025801:Ubiquitin; C464569:USP7 protein, human; D043222:Ubiquitin Thiolesterase; D000076084:Ubiquitin-Specific Peptidase 7