Involvement of histidine in complex formation of PriB and single-stranded DNA.
Involvement of histidine in complex formation of PriB and single-stranded DNA.
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组氨酸参与 PriB 和单链 DNA 的复合物形成。
DOI：
10.1016/j.bbapap.2013.10.015
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发表时间：
2013
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影响因子：
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et al
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作者：
Fujiyama S;et al
PriB 是一种基本的 10 kDa 蛋白，在大肠杆菌中充当 PriA 依赖性复制重启的促进剂。 PriB 具有 OB 折叠二聚体结构，并表现出与单链结合蛋白 (SSB) 类似的单链 DNA (ssDNA) 结合活性。在本研究中，我们使用异核 NMR 分析检查了 PriB 与 ssDNA（oligo-dT35、-dT15 和 -dT7）的相互作用。有趣的是，使用oligo-dT35，PriB 主链的 1H 或 15N 化学位移变化显示出两种不同的模式。主模式中的化学位移扰动位点与先前通过晶体结构分析确定的 PriB-ssDNA 中的主要接触位点一致。结果还表明，ssDNA 中大约 8 nt 是 PriB 的主要接触位点。在二级模式中，α-螺旋区域 (His57-Ser65) 和 β4-loop3-β5 中的残基主要受到干扰。另一方面，我们使用 5'-Cy3- 和 3'-Cy5- 修饰的寡聚 dT35 通过 FRET 检查了 ssDNA 的状态。随着 PriB 浓度的增加，FRET 测定中观察到两步饱和曲线，表明 ssDNA 结构紧凑。此外，我们使用 EMSA 确认了 PriB 与oligo-dT35 的两步结合。 FRET 的 pH 依赖性表明 His 残基的贡献。因此，我们制备了PriB的His突变体，并利用FRET和EMSA发现α螺旋区域的His64促成了PriB和ssDNA之间的第二次相互作用。因此，从结构的角度来看，我们提出了 His64 对 PriB-ssDNA 复合物的致密性和 PriB 的正协同性的作用。
PriB is a basic 10-kDa protein that acts as a facilitator in PriA-dependent replication restart inEscherichia coli. PriB has an OB-fold dimer structure and exhibits single-stranded DNA (ssDNA)-binding activities similar to single-stranded binding protein (SSB). In this study, we examined PriB's interaction with ssDNA (oligo-dT35, -dT15, and -dT7) using heteronuclear NMR analysis. Interestingly,1H or15N chemical shift changes of the PriB main-chain showed two distinct modes using oligo-dT35. The chemical shift perturbation sites in the primary mode were consistent with the main contact site in PriB–ssDNA, which was previously determined by crystal structure analysis. The results also suggested that approximately 8 nt in ssDNA was the main contact site to PriB. In the secondary mode, residues in the α-helix region (His57–Ser65) and in β4–loop3–β5 were mainly perturbed. On the other hand, we examined the state of ssDNA by FRET using 5′-Cy3- and 3′-Cy5-modified oligo-dT35. As the PriB concentration increased, two-step saturation curves were observed in the FRET assay, suggesting a compact structure of ssDNA. Moreover, we confirmed two-step PriB binding to oligo-dT35 using EMSA. The pH dependence of FRET suggested contribution of the His residues. Therefore, we prepared His mutants of PriB and found that His64 in the α-helix region contributed to the second interaction between PriB and ssDNA using FRET and EMSA. Thus, from a structural standpoint, we suggested the role of His64 on the compactness of the PriB–ssDNA complex and on the positive cooperativity of PriB.