5WCH

Crystal structure of the catalytic domain of human USP9X

Summary for 5WCH

• Entry DOI: 10.2210/pdb5wch/pdb
• Descriptor: Probable ubiquitin carboxyl-terminal hydrolase FAF-X, ZINC ION, UNKNOWN ATOM OR ION, ... (4 entities in total)
• Functional Keywords: deubiquitinase, structural genomics, structural genomics consortium, sgc, hydrolase
• Biological source: Homo sapiens (Human)
• Total number of polymer chains: 4
• Total formula weight: 194494.87

Authors

Dong, A.,Zhang, Q.,Walker, J.R.,Bountra, C.,Arrowsmith, C.H.,Edwards, A.M.,Tong, Y.,Structural Genomics Consortium (SGC) (deposition date: 2017-06-30, release date: 2018-07-04, Last modification date: 2024-03-13)

Primary citation

Paudel, P.,Zhang, Q.,Leung, C.,Greenberg, H.C.,Guo, Y.,Chern, Y.H.,Dong, A.,Li, Y.,Vedadi, M.,Zhuang, Z.,Tong, Y.
Crystal structure and activity-based labeling reveal the mechanisms for linkage-specific substrate recognition by deubiquitinase USP9X.
Proc. Natl. Acad. Sci. U.S.A., 116:7288-7297, 2019

PubMed Abstract: 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-Å resolution. The structure reveals a canonical USP-fold comprised of fingers, palm, and thumb subdomains, as well as an unusual β-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 β-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 ABPs form a set of valuable tools to understand polyUb chain processing by the cysteine protease class of DUBs.

PubMed: 30914461
DOI: 10.1073/pnas.1815027116

Experimental method

X-RAY DIFFRACTION (2.5 Å)