title: Molecular domain organization of BldD, an essential transcriptional regulator for developmental process of Streptomyces coelicolor A3(2).
abstract: A homodimeric protein, BldD is a key regulator for developmental process of Streptomyces coelicolor and the bldD mutant exhibits severely pleiotropic defects in the antibiotic production and morphological differentiation of the bacterium. In the present work, we approached domain organization of BldD, to structurally and functionally characterize the protein as a DNA-binding protein. We first observed a proteolytic cleavage of BldD by the cytoplasmic extracts of S. coelicolor, which was highly dependent on the developmental stage of the bacterium. The resulting fragment of BldD was identified by mass spectrometry as the N-terminal domain resistant to the proteolysis. Recombinant proteins corresponding to the intact BldD, the N-terminal domain (residues 1-79) and the rest part (C-terminal domain; residues 80-167) were used for comparative analyses by several spectroscopic, thermodynamic, and biochemical experiments, respectively. The results of circular dichroism and nuclear magnetic resonance spectroscopies certified each of the two determined domains could be regarded as an individual folding unit possessing an independent thermodynamic cooperativity. Structural interaction between the two domains was little observed in the DNA-free and DNA-bound states. Strikingly, it was revealed by gel permeation chromatography, chemical crosslink, gel mobility shift, and NMR-monitored DNA-binding experiments, that only the N-terminal domain is responsible for the dimerization as well as DNA-binding of BldD. Detailed inspection of the present results suggests that BldD function in a unique and complicated mode to totally regulate the diverse developmental stages of S. coelicolor.
authors.affiliation: Laboratory of Biophysics, School of Biological Sciences, and Institute of Microbiology, Seoul National University, Seoul 151-742, Republic of Korea.
authors.forename: Chang-Jin
authors.initials: CJ
authors.lastname: Lee
authors.forename: Hyung-Sik
authors.initials: HS
authors.lastname: Won
authors.forename: Jeong-Mok
authors.initials: JM
authors.lastname: Kim
authors.forename: Bong-Jin
authors.initials: BJ
authors.lastname: Lee
authors.forename: Sa-Ouk
authors.initials: SO
authors.lastname: Kang
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D013058:Mass Spectrometry; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009841:Oligonucleotides / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D048372:Streptomyces coelicolor / Q000737:chemistry*; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C111306:BldD protein, Streptomyces coelicolor; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D014157:Transcription Factors