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

Prajwal Paudel, Qi Zhang, Charles Leung, Harrison C. Greenberg, Yusong Guo, Yi-Hsuan Chern, Aiping Dong, Yanjun Li, Masoud Vedadi, Zhihao Zhuang, Yufeng Tong

Research output: Contribution to journalArticleResearchpeer-review

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.
Original languageEnglish
Pages (from-to)7288-7297
Number of pages10
JournalProceedings of the National Academy of Sciences of the United States of America
Volume116
Issue number15
DOIs
Publication statusPublished - 9 Apr 2019
Externally publishedYes

Keywords

  • deubiquitinase
  • activity-based probes
  • zinc finger
  • linkage specificity
  • USP9X

Cite this

Paudel, Prajwal ; Zhang, Qi ; Leung, Charles ; Greenberg, Harrison C. ; Guo, Yusong ; Chern, Yi-Hsuan ; Dong, Aiping ; Li, Yanjun ; Vedadi, Masoud ; Zhuang, Zhihao ; Tong, Yufeng. / Crystal structure and activity-based labeling reveal the mechanisms for linkage-specific substrate recognition by deubiquitinase USP9X. In: Proceedings of the National Academy of Sciences of the United States of America. 2019 ; Vol. 116, No. 15. pp. 7288-7297.
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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-{\AA} 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.",
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author = "Prajwal Paudel and Qi Zhang and Charles Leung and Greenberg, {Harrison C.} and Yusong Guo and Yi-Hsuan Chern and Aiping Dong and Yanjun Li and Masoud Vedadi and Zhihao Zhuang and Yufeng Tong",
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Crystal structure and activity-based labeling reveal the mechanisms for linkage-specific substrate recognition by deubiquitinase USP9X. / Paudel, Prajwal; Zhang, Qi; Leung, Charles ; Greenberg, Harrison C.; Guo, Yusong; Chern, Yi-Hsuan; Dong, Aiping; Li, Yanjun; Vedadi, Masoud; Zhuang, Zhihao; Tong, Yufeng.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 116, No. 15, 09.04.2019, p. 7288-7297.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Crystal structure and activity-based labeling reveal the mechanisms for linkage-specific substrate recognition by deubiquitinase USP9X

AU - Paudel, Prajwal

AU - Zhang, Qi

AU - Leung, Charles

AU - Greenberg, Harrison C.

AU - Guo, Yusong

AU - Chern, Yi-Hsuan

AU - Dong, Aiping

AU - Li, Yanjun

AU - Vedadi, Masoud

AU - Zhuang, Zhihao

AU - Tong, Yufeng

PY - 2019/4/9

Y1 - 2019/4/9

N2 - 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.

AB - 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.

KW - deubiquitinase

KW - activity-based probes

KW - zinc finger

KW - linkage specificity

KW - USP9X

U2 - 10.1073/pnas.1815027116

DO - 10.1073/pnas.1815027116

M3 - Article

VL - 116

SP - 7288

EP - 7297

JO - Proceedings of the National Academy of Sciences

JF - Proceedings of the National Academy of Sciences

SN - 0027-8424

IS - 15

ER -