Identification of Serine 119 as an Effective Inhibitor Binding Site of M. tuberculosis Ubiquitin-like Protein Ligase PafA Using Purified Proteins and M. smegmatis
He-Wei Jiang,
Daniel M. Czajkowsky,
Tao Wang,
Xu-De Wang,
Jia-bin Wang,
Hai-Nan Zhang,
Cheng-Xi Liu,
Fan-Lin Wu,
Xiang He,
Zhao-Wei Xu,
Hong Chen,
Shu-Juan Guo,
Yang Li,
Li-Jun Bi,
Jiao-Yu Deng,
Jin Xie,
Jian-Feng Pei,
Xian-En Zhang,
Sheng-Ce Tao
Affiliations
He-Wei Jiang
Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China
Daniel M. Czajkowsky
School of Biomedical Engineering, Bio-ID Center, Shanghai Jiao Tong University, Shanghai 200240, China; School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China
Tao Wang
Department of Biology, Southern University of Science and Technology, Shenzhen 518055, China; SZCDC-SUSTech Joint Key Laboratory for Tropical Diseases, Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China
Xu-De Wang
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
Jia-bin Wang
Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China
Hai-Nan Zhang
Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China
Cheng-Xi Liu
Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China
Fan-Lin Wu
Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China
Xiang He
Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China
Zhao-Wei Xu
Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China
Hong Chen
Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China
Shu-Juan Guo
Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China
Yang Li
Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China
Li-Jun Bi
National Key Laboratory of Biomacromolecules, Key Laboratory of Non-Coding RNA and Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; TB Healthcare Biotechnology Co., Ltd., Foshan, Guangdong 528000, China; School of Stomatology and Medicine, Foshan University, Foshan 528000, Guangdong Province, China
Jiao-Yu Deng
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
Jin Xie
Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
Jian-Feng Pei
Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
Xian-En Zhang
National Key Laboratory of Biomacromolecules, Key Laboratory of Non-Coding RNA and Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
Sheng-Ce Tao
Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China; School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University, Shanghai 200240, China; Corresponding author at: Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200240, China.
Owing to the spread of multidrug resistance (MDR) and extensive drug resistance (XDR), there is a pressing need to identify potential targets for the development of more-effective anti-M. tuberculosis (Mtb) drugs. PafA, as the sole Prokaryotic Ubiquitin-like Protein ligase in the Pup-proteasome System (PPS) of Mtb, is an attractive drug target. Here, we show that the activity of purified Mtb PafA is significantly inhibited upon the association of AEBSF (4-(2-aminoethyl) benzenesulfonyl fluoride) to PafA residue Serine 119 (S119). Mutation of S119 to amino acids that resemble AEBSF has similar inhibitory effects on the activity of purified Mtb PafA. Structural analysis reveals that although S119 is distant from the PafA catalytic site, it is located at a critical position in the groove where PafA binds the C-terminal region of Pup. Phenotypic studies demonstrate that S119 plays critical roles in the function of Mtb PafA when tested in M. smegmatis. Our study suggests that targeting S119 is a promising direction for developing an inhibitor of M. tuberculosis PafA. Keywords: M. Tuberculosis, Pupylation, PafA, 4-(2-aminoethyl) benzenesulfonyl fluoride
