title: Characterization of amino acid residues essential for tetramer formation and DNA-binding activity of ssDNA-binding protein of Mycobacterium tuberculosis.
abstract: Mycobacterium tuberculosis Rv0054 encodes a single-stranded DNA-binding protein (MtbSSB) that is essential for survival of the human pathogen and causative agent of tuberculosis. The function of MtbSSB has been proposed to be different from its E. coli homolog. However, the critical amino acid residues of MtbSSB and their regulatory effects on DNA-binding ability remain to be clearly characterized. In this study, using a frequency-controlled random mutagenesis method (FRM), mutant libraries of MtbSSB were successfully constructed. On the whole, 146 single, double, and triple MtbSSB mutants, which covered 89% of the amino acid residues along the whole MtbSSB gene, were isolated. Using bacterial two-hybrid assays in combination with native PAGE assays, four new mutants, E62G, D104N, E94G/T137N, and S130P/G153N were found to totally or partially lose their ability to form tetramer. Three novel mutants, E62G, D104N, and E94G/T134N, were characterized to have a much lower ssDNA-binding activity, while one mutant, F21L, was found to have a significantly higher activity through both electrophoretic mobility shift and surface plasmon resonance assays. Interestingly, three amino acid residues, E62, D104, and E94, were found to regulate both oligomerization and ssDNA-binding activity of MtbSSB. Our work provides an important resource and should help improve the understanding of the biochemical mechanisms and structure-function relationship of the DNA-binding protein in this important human pathogen.
authors.affiliation: National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Zhang
authors.forename: Yuxi
authors.initials: Y
authors.lastname: Tian
authors.forename: Ziduo
authors.initials: Z
authors.lastname: Liu
authors.forename: Feng
authors.initials: F
authors.lastname: Huang
authors.forename: Lihua
authors.initials: L
authors.lastname: Hu
authors.forename: Zheng-Guo
authors.initials: ZG
authors.lastname: He
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins