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Proc Natl Acad Sci U S A. 2011 May 10;108(19):7751-6. doi: 10.1073/pnas.1102294108. Epub 2011 Apr 25.

Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design.

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  • 1Department of Biochemistry and Molecular Biology, University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA.

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.

PMID:
21518904
[PubMed - indexed for MEDLINE]
PMCID:
PMC3093456
Free PMC Article
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