Inactivity of YGL082W in vitro due to impairment of conformational change in the catalytic center loop  

作　　者：Lining Lu Yu Guo Tian Wang Lujun Liang Suwen Zhao Feng Wang Lei Liu 

机构地区：[1]Tsinghua-Peking Center for Life Sciences,Key Laboratory of Bioorganic Phosphorus Chemistry&Chemical Biology(Ministry of Education),Center for Synthetic and Systems Biology,State Key Laboratory of Chemical Oncogenomics(Shenzhen),Department of Chemistry,Tsinghua University,Beijing 100084,China [2]School of Life Science,Beijing Institute of Technology,Beijing 100081,China [3]iHuman Institute,School of Life Science and Technology,ShanghaiTech University,Shanghai 201210,China

出　　处：《Science China Chemistry》2020年第2期237-243,共7页中国科学（化学英文版）

基　　金：supported by the National Key Research and Development Program of China(2017YFA0505200);the National Natural Science Foundation of China(21532004,91753205,81621002,21621003);Shanghai Tech University

摘　　要：MINDY-1 is a recently discovered new family of deubiquitinating enzymes(DUB),but one of its yeast homologs,YGL082 W,does not show any DUB activity in vitro.Sequence alignment shows that YGL082 W possesses the correct catalytic triad,and yet did not catalyze either the hydrolysis of di-ubiquitin,crosslinking with C-terminally propargylated ubiquitin,or hydrolysis of ubiquitin-7-amino-4-methylcoumarin.After obtaining a crystal structure of the catalytic domain of YGL082 W,we identified an interesting difference between the catalytic center loop of YGL082 W and that of its human homolog MINDY-1.Because the conformation of the catalytic center loop was previously reported to be important for the deubiquitination activity of MINDY-1,we hypothesized that Glu27(instead of the corresponding Pro136 in MINDY-1) of the catalytic center loop of YGL082 W may impair the conformational change and account for the lack of activity.This hypothesis was supported by homology modeling and molecular dynamics simulations,which showed that the Pro-to-Glu mutation(P136 E mutation for MINDY-1) creates a hydrogen bond that inhibits the conformation change of the catalytic center loop of MINDY-1.Further experiments through site-directed mutation validated this hypothesis,showing that the P27 E mutation caused MIY1(a homologous active DUB from yeast) to lose activity.MINDY-1 is a recently discovered new family of deubiquitinating enzymes(DUB),but one of its yeast homologs,YGL082 W,does not show any DUB activity in vitro.Sequence alignment shows that YGL082 W possesses the correct catalytic triad,and yet did not catalyze either the hydrolysis of di-ubiquitin,crosslinking with C-terminally propargylated ubiquitin,or hydrolysis of ubiquitin-7-amino-4-methylcoumarin.After obtaining a crystal structure of the catalytic domain of YGL082 W,we identified an interesting difference between the catalytic center loop of YGL082 W and that of its human homolog MINDY-1.Because the conformation of the catalytic center loop was previously reported to be important for the deubiquitination activity of MINDY-1,we hypothesized that Glu27(instead of the corresponding Pro136 in MINDY-1) of the catalytic center loop of YGL082 W may impair the conformational change and account for the lack of activity.This hypothesis was supported by homology modeling and molecular dynamics simulations,which showed that the Pro-to-Glu mutation(P136 E mutation for MINDY-1) creates a hydrogen bond that inhibits the conformation change of the catalytic center loop of MINDY-1.Further experiments through site-directed mutation validated this hypothesis,showing that the P27 E mutation caused MIY1(a homologous active DUB from yeast) to lose activity.

关 键 词：deubiquitinating enzymes(DUB) ENZYMOLOGY X-ray crystal structure molecular dynamics 

分 类 号：O643.361[理学—物理化学]