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

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

Research output: Contribution to journalArticleResearchpeer-review

22 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2750-2757
Number of pages8
JournalNucleic Acids Research
Volume42
Issue number4
DOIs
Publication statusPublished - Feb 2014

Cite this

Shishmarev, Dmitry ; Wang, Yao ; Mason, Claire E. ; Su, Xun-Cheng ; Oakley, Aaron J ; Graham, Bim ; Huber, Thomas ; Dixon, Nicholas Edward ; Otting, Gottfried. / Intramolecular binding mode of the C-terminus of Escherichia coli single-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy. In: Nucleic Acids Research. 2014 ; Vol. 42, No. 4. pp. 2750-2757.
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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.",
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Shishmarev, D, Wang, Y, Mason, CE, Su, X-C, Oakley, AJ, Graham, B, Huber, T, Dixon, NE & 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, vol. 42, no. 4, pp. 2750-2757. https://doi.org/10.1093/nar/gkt1238

Intramolecular binding mode of the C-terminus of Escherichia coli single-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy. / Shishmarev, Dmitry; Wang, Yao; Mason, Claire E.; Su, Xun-Cheng; Oakley, Aaron J; Graham, Bim; Huber, Thomas; Dixon, Nicholas Edward; Otting, Gottfried.

In: Nucleic Acids Research, Vol. 42, No. 4, 02.2014, p. 2750-2757.

Research output: Contribution to journalArticleResearchpeer-review

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AU - Shishmarev, Dmitry

AU - Wang, Yao

AU - Mason, Claire E.

AU - Su, Xun-Cheng

AU - Oakley, Aaron J

AU - Graham, Bim

AU - Huber, Thomas

AU - Dixon, Nicholas Edward

AU - Otting, Gottfried

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N2 - 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.

AB - 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.

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M3 - Article

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