title: Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design.
abstract: Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a "monobody," a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserved SIM-binding site while discriminating individual SUMO isoforms. Based on this structure, we designed a SUMO-targeted library from which we obtained monobodies that bound to the SIM-binding site of human SUMO1 with K(d) values of approximately 100 nM but bound to SUMO2 400 times more weakly. The monobodies inhibited SUMO1/SIM interactions and, unexpectedly, also inhibited SUMO1 conjugation. These high-affinity and isoform-specific inhibitors will enhance mechanistic and cellular investigations of SUMO biology.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA.
authors.forename: Ryan N
authors.initials: RN
authors.lastname: Gilbreth
authors.forename: Khue
authors.initials: K
authors.lastname: Truong
authors.forename: Ikenna
authors.initials: I
authors.lastname: Madu
authors.forename: Akiko
authors.initials: A
authors.lastname: Koide
authors.forename: John B
authors.initials: JB
authors.lastname: Wojcik
authors.forename: Nan-Sheng
authors.initials: NS
authors.lastname: Li
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Piccirilli
authors.forename: Yuan
authors.initials: Y
authors.lastname: Chen
authors.forename: Shohei
authors.initials: S
authors.lastname: Koide
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D015195:Drug Design; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D025842:SUMO-1 Protein / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D014452:Ubiquitins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism
chemical_list: D019151:Peptide Library; D010455:Peptides; D020033:Protein Isoforms; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; C117909:SUMO2 protein, human; C492189:SUMO3 protein, human; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D014452:Ubiquitins