Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 108, Issue 19, Pages 7751-7756Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1102294108
Keywords
protein engineering; molecular recognition; posttranslational modification; antibody mimic; fibronectin type III domain
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Funding
- National Institutes of Health [R01-GM72688, R01-GM090324, R21-CA132700]
- University of Chicago Cancer Research Center
- Michigan Economic Development Corporation
- Michigan Technology Tri-Corridor [085P1000817]
- US Department of Energy, Office of Science, Office of Basic Energy Sciences [DE-AC02-06CH11357]
- [T32 GM007183-32A1]
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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.
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