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

Science China Chemistry volume 63, pages 237–243 (2020)Cite this article

Abstract

MINDY-1 is a recently discovered new family of deubiquitinating enzymes (DUB), but one of its yeast homologs, YGL082W, does not show any DUB activity in vitro. Sequence alignment shows that YGL082W 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 YGL082W, we identified an interesting difference between the catalytic center loop of YGL082W 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 YGL082W 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 (P136E 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 P27E mutation caused MIY1 (a homologous active DUB from yeast) to lose activity.

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Acknowledgements

The work was supported by the National Key Research and Development Program of China (2017YFA0505200), the National Natural Science Foundation of China (21532004, 91753205, 81621002, 21621003) and ShanghaiTech University.

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Authors and Affiliations

  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

    Lining Lu, Tian Wang, Lujun Liang & Lei Liu

  2. School of Life Science, Beijing Institute of Technology, Beijing, 100081, China

    Feng Wang

  3. Human Institute, School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China

    Yu Guo & Suwen Zhao

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  1. Lining Lu
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  2. Yu Guo
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  3. Tian Wang
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  4. Lujun Liang
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  5. Suwen Zhao
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  6. Feng Wang
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  7. Lei Liu
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Correspondence to Suwen Zhao, Feng Wang or Lei Liu.

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Lu, L., Guo, Y., Wang, T. et al. Inactivity of YGL082W in vitro due to impairment of conformational change in the catalytic center loop. Sci. China Chem. 63, 237–243 (2020). https://doi.org/10.1007/s11426-019-9623-0

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  • DOI: https://doi.org/10.1007/s11426-019-9623-0

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