TY - JOUR
T1 - Identification and structure-function analyses of an allosteric inhibitor of the tyrosine phosphatase PTPN22
AU - Li, Kangshuai
AU - Hou, Xuben
AU - Li, Ruirui
AU - Bi, Wenxiang
AU - Yang, Fan
AU - Chen, Xu
AU - Xiao, Peng
AU - Liu, Tiantian
AU - Lu, Tiange
AU - Zhou, Yuan
AU - Tian, Zhaomei
AU - Shen, Yuemao
AU - Zhang, Yingkai
AU - Wang, Jiangyun
AU - Fang, Hao
AU - Sun, Jinpeng
AU - Yu, Xiao
N1 - Publisher Copyright:
© 2019 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved.
PY - 2019/5/24
Y1 - 2019/5/24
N2 - Protein-tyrosine phosphatase nonreceptor type 22 (PTPN22) is a lymphoid-specific tyrosine phosphatase (LYP), and mutations in the PTPN22 gene are highly correlated with a spectrum of autoimmune diseases. However, compounds and mechanisms that specifically inhibit LYP enzymes to address therapeutic needs to manage these diseases remain to be discovered. Here, we conducted a similarity search of a commercial database for PTPN22 inhibitors and identified several LYP inhibitor scaffolds, which helped identify one highly active inhibitor, NC1. Using noncompetitive inhibition curve and phosphatase assays, we determined NC1's inhibition mode toward PTPN22 and its selectivity toward a panel of phosphatases.Wefound thatNC1is a noncompetitive LYP inhibitor and observed that it exhibits selectivity against other protein phosphatases and effectively inhibits LYP activity in lymphoid T cells and modulates T-cell receptor signaling. Results from site-directed mutagenesis, fragment- centric topographic mapping, and molecular dynamics simulation experiments suggested that NC1, unlike other known LYP inhibitors, concurrently binds to a "WPD" pocket and a second pocket surrounded by an LYP-specific insert, which contributes to its selectivity against other phosphatases. Moreover, using a newly developed method to incorporate the unnatural amino acid 2-fluorine-tyrosine and 19F NMR spectroscopy, we provide direct evidence that NC1 allosterically regulates LYP activity by restricting WPD-loop movement. In conclusion, our approach has identified a new allosteric binding site in LYP useful for selective LYP inhibitor development; we propose that the 19F NMR probe developed here may also be useful for characterizing allosteric inhibitors of other tyrosine phosphatases.
AB - Protein-tyrosine phosphatase nonreceptor type 22 (PTPN22) is a lymphoid-specific tyrosine phosphatase (LYP), and mutations in the PTPN22 gene are highly correlated with a spectrum of autoimmune diseases. However, compounds and mechanisms that specifically inhibit LYP enzymes to address therapeutic needs to manage these diseases remain to be discovered. Here, we conducted a similarity search of a commercial database for PTPN22 inhibitors and identified several LYP inhibitor scaffolds, which helped identify one highly active inhibitor, NC1. Using noncompetitive inhibition curve and phosphatase assays, we determined NC1's inhibition mode toward PTPN22 and its selectivity toward a panel of phosphatases.Wefound thatNC1is a noncompetitive LYP inhibitor and observed that it exhibits selectivity against other protein phosphatases and effectively inhibits LYP activity in lymphoid T cells and modulates T-cell receptor signaling. Results from site-directed mutagenesis, fragment- centric topographic mapping, and molecular dynamics simulation experiments suggested that NC1, unlike other known LYP inhibitors, concurrently binds to a "WPD" pocket and a second pocket surrounded by an LYP-specific insert, which contributes to its selectivity against other phosphatases. Moreover, using a newly developed method to incorporate the unnatural amino acid 2-fluorine-tyrosine and 19F NMR spectroscopy, we provide direct evidence that NC1 allosterically regulates LYP activity by restricting WPD-loop movement. In conclusion, our approach has identified a new allosteric binding site in LYP useful for selective LYP inhibitor development; we propose that the 19F NMR probe developed here may also be useful for characterizing allosteric inhibitors of other tyrosine phosphatases.
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U2 - 10.1074/jbc.RA118.007129
DO - 10.1074/jbc.RA118.007129
M3 - Article
C2 - 30979725
AN - SCOPUS:85066429484
SN - 0021-9258
VL - 294
SP - 8653
EP - 8663
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 21
ER -