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.
Nucleic Acids Res. 2014 Feb;42(4):2750-7. doi: 10.1093/nar/gkt1238. Epub 2013 Nov 27.
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.
单链 DNA（ssDNA）结合蛋白（SSB）是一种保护 ssDNA 并招募特定 ssDNA 加工蛋白的必需蛋白。大肠杆菌 SSB 在中性 pH 下形成四聚体，由结构上定义明确的 ssDNA 结合结构域（OB 结构域）和大约 64 个氨基酸残基的无序 C 末端结构域（C 结构域）组成。SSB 的 C 末端 8 个残基片段（C 肽）已被证明与 OB 结构域相互作用，但晶体结构未能显示 C 肽的任何电子密度。在这里，我们表明 SSB 在 pH 值为 3.4 时形成单体，这适合用于高分辨率核磁共振（NMR）光谱研究。单体中的 OB 结构域保留其 3D 结构，并且 C 肽通过核 Overhauser 效应和镧系元素诱导的假接触位移显示与晶体结构中 SSB-ssDNA 复合物中的 OB 结构域结合，在该晶体结构中，OB 结构域结合在 ssDNA 上。（15）N 弛豫数据表明，连接 C 肽和 OB 结构域的多肽片段具有很高的灵活性，与 OB 结构域相比，C 肽的灵活性略有增加，这表明 C 肽在结合状态下保持高迁移率，或者处于与未结合状态的快速平衡状态。
