title: Intramolecular binding mode of the C-terminus of Escherichia coli single-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy.
abstract: Single-stranded DNA (ssDNA) binding protein (SSB) is an essential protein to protect ssDNA and recruit specific ssDNA-processing proteins. Escherichia coli SSB forms a tetramer at neutral pH, comprising a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain) of ∼ 64 amino acid residues. The C-terminal eight-residue segment of SSB (C-peptide) has been shown to interact with the OB-domain, but crystal structures failed to reveal any electron density of the C-peptide. Here we show that SSB forms a monomer at pH 3.4, which is suitable for studies by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The OB-domain retains its 3D structure in the monomer, and the C-peptide is shown by nuclear Overhauser effects and lanthanide-induced pseudocontact shifts to bind to the OB-domain at a site that harbors ssDNA in the crystal structure of the SSB-ssDNA complex. (15)N relaxation data demonstrate high flexibility of the polypeptide segment linking the C-peptide to the OB-domain and somewhat increased flexibility of the C-peptide compared with the OB-domain, suggesting that the C-peptide either retains high mobility in the bound state or is in a fast equilibrium with an unbound state.
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory 0200, Australia, Centre for Medical and Molecular Bioscience and School of Chemistry, University of Wollongong, New South Wales 2522, Australia and Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Parkville Victoria 3052, Australia.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Shishmarev
authors.forename: Yao
authors.initials: Y
authors.lastname: Wang
authors.forename: Claire E
authors.initials: CE
authors.lastname: Mason
authors.forename: Xun-Cheng
authors.initials: XC
authors.lastname: Su
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Oakley
authors.forename: Bim
authors.initials: B
authors.lastname: Graham
authors.forename: Thomas
authors.initials: T
authors.lastname: Huber
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Dixon
authors.forename: Gottfried
authors.initials: G
authors.lastname: Otting
mesh_terms: D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D010455:Peptides; C476430:SSB protein, E coli