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Intramolecular binding mode of the C-terminus of Escherichia coli single-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy

Journal Article


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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. 15N 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


  •   Shishmarev, Dmitry (external author)
  •   Wang, Yao (external author)
  •   Mason, Claire (external author)
  •   Su, Xun-Cheng (external author)
  •   Oakley, Aaron J.
  •   Graham, Bim (external author)
  •   Huber, Thomas (external author)
  •   Dixon, Nicholas E.
  •   Otting, Gottfried (external author)

Publication Date


  • 2014

Citation


  • Shishmarev, D., Wang, Y., Mason, C. E., Su, X., Oakley, A. J., Graham, B., Huber, T., Dixon, N. E. & Otting, G. (2014). Intramolecular binding mode of the C-terminus of Escherichia coli single-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy. Nucleic Acids Research, 42 (4), 2750-2757.

Scopus Eid


  • 2-s2.0-84895822845

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=2577&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/1559

Number Of Pages


  • 7

Start Page


  • 2750

End Page


  • 2757

Volume


  • 42

Issue


  • 4

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. 15N 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


  •   Shishmarev, Dmitry (external author)
  •   Wang, Yao (external author)
  •   Mason, Claire (external author)
  •   Su, Xun-Cheng (external author)
  •   Oakley, Aaron J.
  •   Graham, Bim (external author)
  •   Huber, Thomas (external author)
  •   Dixon, Nicholas E.
  •   Otting, Gottfried (external author)

Publication Date


  • 2014

Citation


  • Shishmarev, D., Wang, Y., Mason, C. E., Su, X., Oakley, A. J., Graham, B., Huber, T., Dixon, N. E. & Otting, G. (2014). Intramolecular binding mode of the C-terminus of Escherichia coli single-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy. Nucleic Acids Research, 42 (4), 2750-2757.

Scopus Eid


  • 2-s2.0-84895822845

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=2577&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/1559

Number Of Pages


  • 7

Start Page


  • 2750

End Page


  • 2757

Volume


  • 42

Issue


  • 4