文献类型：期刊文献

英文题名：Crystal structure and activity-based labeling reveal the mechanisms for linkage-specific substrate recognition by deubiquitinase USP9X

作者：Paudel, Prajwal[1];Zhang, Qi[2];Leung, Charles[2];Greenberg, Harrison C.[1];Guo, Yusong[3];Chern, Yi-Hsuan[4];Dong, Aiping[2];Li, Yanjun[2];Vedadi, Masoud[2,4];Zhuang, Zhihao[1];Tong, Yufeng[2,4,5]

机构：[1]Univ Delaware, Dept Chem & Biochem, Newark, DE 19716 USA;[2]Univ Toronto, Struct Genom Consortium, Toronto, ON M5G 1L7, Canada;[3]Guangdong Ocean Univ, Fisheries Coll, Zhanjiang 524025, Guangdong, Peoples R China;[4]Univ Toronto, Dept Pharmacol & Toxicol, Toronto, ON M5S 1A8, Canada;[5]Univ Windsor, Dept Chem & Biochem, Windsor, ON N9B 3P4, Canada

年份：2019

卷号：116

期号：15

起止页码：7288

外文期刊名：PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA

收录：SCI-EXPANDED(收录号：WOS:000463936900025)、、WOS

基金：The Structural Genomics Consortium is a United Kingdom registered charity (no. 1097737) that receives funds from AbbVie, Bayer, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada through the Ontario Genomics Institute (OGI-055), Innovative Medicines Initiative (EU/EFPIA) (ULTRA-DD Grant 115766), Janssen, Merck KGaA, MSD, Novartis, Ontario Ministry of Research, Innovation and Science, Pfizer, Sao Paulo Research Foundation-FAPESP, Takeda, and the Wellcome Trust. X-ray data were collected at the Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source, which is operated by the University of Chicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research under Contract DE-AC02-06CH11357. This work was supported in part by National Institutes of Health Grants R01GM097468 and R21NS085509 (to Z.Z.); National Sciences and Engineering Research Council of Canada Grant RGPIN-2017-06520 (to Y.T.); and a joint Janssen-Ontario Centre of Excellences Neuroscience Catalyst grant (to Y.T. and M.V.). It was also supported by the Delaware COBRE program for instrumentation facilities, with a grant from the National Institute of General Medical Sciences (1 P30 GM110758-01). Y.G. is a visiting scholar sponsored by the Excellent Young Teachers program and Innovation Fund of Guangdong Ocean University.

语种：英文

外文关键词：deubiquitinase; activity-based probes; zinc finger; linkage specificity; USP9X

外文摘要：USP9X is a conserved deubiquitinase (DUB) that regulates multiple cellular processes. Dysregulation of USP9X has been linked to cancers and X-linked intellectual disability. Here, we report the crystal structure of the USP9X catalytic domain at 2.5-angstrom resolution. The structure reveals a canonical USP-fold comprised of fingers, palm, and thumb subdomains, as well as an unusual beta-hairpin insertion. The catalytic triad of USP9X is aligned in an active configuration. USP9X is exclusively active against ubiquitin (Ub) but not Ub-like modifiers. Cleavage assays with di-, tri-, and tetraUb chains show that the USP9X catalytic domain has a clear preference for K11-, followed by K63-, K48-, and K6-linked polyUb chains. Using a set of activity-based diUb and triUb probes (ABPs), we demonstrate that the USP9X catalytic domain has an exo-cleavage preference for K48- and endo-cleavage preference for K11-linked polyUb chains. The structure model and biochemical data suggest that the USP9X catalytic domain harbors three Ub binding sites, and a zinc finger in the fingers subdomain and the p-hairpin insertion both play important roles in polyUb chain processing and linkage specificity. Furthermore, unexpected labeling of a secondary, noncatalytic cysteine located on a blocking loop adjacent to the catalytic site by K11-diUb ABP implicates a previously unreported mechanism of polyUb chain recognition. The structural features of USP9X revealed in our study are critical for understanding its DUB activity. The new Ub-based ABP5 form a set of valuable tools to understand polyUb chain processing by the cysteine protease class of DUBs.