title: Identification of the DNA binding surface of H-NS protein from Escherichia coli by heteronuclear NMR spectroscopy.
abstract: The DNA binding domain of H-NS protein was studied with various N-terminal deletion mutant proteins and identified by gel retardation assay and heteronuclear 2D- and 3D-NMR spectroscopies. It was shown from gel retardation assay that DNA binding affinity of the mutant proteins relative to that of native H-NS falls in the range from 1/6 to 1/25 for H-NS(60-137), H-NS(70-137) and H-NS(80-137), whereas it was much weaker for H-NS(91-137). Thus, the DNA binding domain was defined to be the region from residue A80 to the C-terminus. Sequential nuclear Overhauser effect (NOE) connectivities and those of medium ranges revealed that the region of residues Q60-R93 in mutant protein H-NS(60-137) forms a long stretch of disordered, flexible chain, and also showed that the structure of the C-terminal region (residues A95-Q137) in mutant H-NS(60-137) was nearly identical to that of H-NS(91-137). 1H and 15N chemical shift perturbations induced by complex formation of H-NS(60-137) with an oligonucleotide duplex 14-mer demonstrated that two loop regions, i.e. residues A80-K96 and T110-A117, play an essential role in DNA binding.
authors.affiliation: School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Japan. shindo@ps.toyaku.ac.jp
authors.forename: H
authors.initials: H
authors.lastname: Shindo
authors.forename: A
authors.initials: A
authors.lastname: Ohnuki
authors.forename: H
authors.initials: H
authors.lastname: Ginba
authors.forename: E
authors.initials: E
authors.lastname: Katoh
authors.forename: C
authors.initials: C
authors.lastname: Ueguchi
authors.forename: T
authors.initials: T
authors.lastname: Mizuno
authors.forename: T
authors.initials: T
authors.lastname: Yamazaki
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; D004247:DNA