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The Conserved Lys-95 Charged Residue Cluster Is Critical for the Homodimerization and Enzyme Activity of Human Ribonucleotide Reductase Small Subunit M2

文献类型：期刊论文

作者Chen, Xinhuan1; Xu, Zhijian3; Zhang, Lingna1; Liu, Hongchuan3; Liu, Xia1; Lou, Meng1; Zhu, Lijun1; Huang, Bingding2; Yang, Cai-Guang3; Zhu, Weiliang3
刊名JOURNAL OF BIOLOGICAL CHEMISTRY
出版日期2014-01-10
卷号289期号:2页码:909-920
ISSN号0021-9258
DOI10.1074/jbc.M113.524546
文献子类Article
英文摘要Ribonucleotide reductase (RR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides for DNA synthesis. Human RR small subunit M2 exists in a homodimer form. However, the importance of the dimer form to the enzyme and the related mechanism remain unclear. In this study, we tried to identify the interfacial residues that may mediate the assembly of M2 homodimer by computational alanine scanning based on the x-ray crystal structure. Co-immunoprecipitation, size exclusion chromatography, and RR activity assays showed that the K95E mutation in M2 resulted in dimer disassembly and enzyme activity inhibition. In comparison, the charge-exchanging double mutation of K95E and E98K recovered the dimerization and activity. Structural comparisons suggested that a conserved cluster of charged residues, including Lys-95, Glu-98, Glu-105, and Glu-174, at the interface may function as an ionic lock for M2 homodimer. Although the measurements of the radical and iron contents showed that the monomer (the K95E mutant) was capable of generating the diiron and tyrosyl radical cofactor, co-immunoprecipitation and competitive enzyme inhibition assays indicated that the disassembly of M2 dimer reduced its interaction with the large subunit M1. In addition, the immunofluorescent and fusion protein-fluorescent imaging analyses showed that the dissociation of M2 dimer altered its subcellular localization. Finally, the transfection of the wild-type M2 but not the K95E mutant rescued the G(1)/S phase cell cycle arrest and cell growth inhibition caused by the siRNA knockdown of M2. Thus, the conserved Lys-95 charged residue cluster is critical for human RR M2 homodimerization, which is indispensable to constitute an active holoenzyme and function in cells.
WOS关键词PARTICLE MESH EWALD ; ESCHERICHIA-COLI ; IN-VITRO ; PROTEIN R2 ; SUBCELLULAR-LOCALIZATION ; SACCHAROMYCES-CEREVISIAE ; ALLOSTERIC REGULATION ; MOLECULAR-MECHANICS ; ELECTRON-TRANSFER ; DNA-REPLICATION
资助项目National Natural Science Foundation of China[81372138] ; National Natural Science Foundation of China[30873094] ; National Natural Science Foundation of China[81090421] ; 863 National High Technology Research and Development Program of China[2012AA020206] ; 863 National High Technology Research and Development Program of China[2012AA01A305] ; Ministry of Education of China[J20100041]
WOS研究方向Biochemistry & Molecular Biology
语种英语
出版者AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
WOS记录号WOS:000330541200026
源URL[http://119.78.100.183/handle/2S10ELR8/277232]  
专题药物发现与设计中心
通讯作者Zhu, Weiliang
作者单位1.Zhejiang Univ, Dept Pathol & Pathophysiol, Sch Med, Hangzhou 310058, Zhejiang, Peoples R China;
2.German Canc Res Ctr, Genom & Prote Core Facil, D-69120 Heidelberg, Germany
3.Chinese Acad Sci, Shanghai Inst Mat Med, Shanghai 201203, Peoples R China;
推荐引用方式
GB/T 7714		 Chen, Xinhuan,Xu, Zhijian,Zhang, Lingna,et al. The Conserved Lys-95 Charged Residue Cluster Is Critical for the Homodimerization and Enzyme Activity of Human Ribonucleotide Reductase Small Subunit M2[J]. JOURNAL OF BIOLOGICAL CHEMISTRY,2014,289(2):909-920.
APA Chen, Xinhuan.,Xu, Zhijian.,Zhang, Lingna.,Liu, Hongchuan.,Liu, Xia.,...&Shao, Jimin.(2014).The Conserved Lys-95 Charged Residue Cluster Is Critical for the Homodimerization and Enzyme Activity of Human Ribonucleotide Reductase Small Subunit M2.JOURNAL OF BIOLOGICAL CHEMISTRY,289(2),909-920.
MLA Chen, Xinhuan,et al."The Conserved Lys-95 Charged Residue Cluster Is Critical for the Homodimerization and Enzyme Activity of Human Ribonucleotide Reductase Small Subunit M2".JOURNAL OF BIOLOGICAL CHEMISTRY 289.2(2014):909-920.

入库方式： OAI收割

来源：上海药物研究所

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