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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{sub 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 enhancemore » mechanistic and cellular investigations of SUMO biology.« less

Authors:
; ; ; ; ; ; ; ;  [1];  [2]
  1. (UC)
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
UNIVERSITYNIH
OSTI Identifier:
1020580
Resource Type:
Journal Article
Journal Name:
Proc. Natl. Acad. Sci. USA
Additional Journal Information:
Journal Volume: 108; Journal Issue: (19) ; 05, 2011; Journal ID: ISSN 0027-8424
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; BIOLOGY; CRYSTAL STRUCTURE; DESIGN; FUNCTIONALS; MODIFICATIONS; PROTEIN ENGINEERING; PROTEINS; REGULATIONS; YEASTS

Citation Formats

Gilbreth, Ryan N., Truong, Khue, Madu, Ikenna, Koide, Akiko, Wojcik, John B., Li, Nan-Sheng, Piccirilli, Joseph A., Chen, Yuan, Koide, Shohei, and CHNMC). Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design. United States: N. p., 2011. Web. doi:10.1073/pnas.1102294108.
Gilbreth, Ryan N., Truong, Khue, Madu, Ikenna, Koide, Akiko, Wojcik, John B., Li, Nan-Sheng, Piccirilli, Joseph A., Chen, Yuan, Koide, Shohei, & CHNMC). Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design. United States. doi:10.1073/pnas.1102294108.
Gilbreth, Ryan N., Truong, Khue, Madu, Ikenna, Koide, Akiko, Wojcik, John B., Li, Nan-Sheng, Piccirilli, Joseph A., Chen, Yuan, Koide, Shohei, and CHNMC). Mon . "Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design". United States. doi:10.1073/pnas.1102294108.
@article{osti_1020580,
title = {Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design},
author = {Gilbreth, Ryan N. and Truong, Khue and Madu, Ikenna and Koide, Akiko and Wojcik, John B. and Li, Nan-Sheng and Piccirilli, Joseph A. and Chen, Yuan and Koide, Shohei and CHNMC)},
abstractNote = {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{sub 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.},
doi = {10.1073/pnas.1102294108},
journal = {Proc. Natl. Acad. Sci. USA},
issn = {0027-8424},
number = (19) ; 05, 2011,
volume = 108,
place = {United States},
year = {2011},
month = {7}
}