Huang Yen-Hua, Guan Hong-Hsiang, Chen Chun-Jung, Huang Cheng-Yang
School of Biomedical Sciences, Chung Shan Medical University, Taichung City, Taiwan.
Life Science Group, Scientific Research Division, National Synchrotron Radiation Research Center, Hsinchu, Taiwan.
PLoS One. 2017 Jul 27;12(7):e0182060. doi: 10.1371/journal.pone.0182060. eCollection 2017.
Single-stranded DNA-binding protein (SSB) and PriA helicase play important roles in bacterial DNA replication restart process. The mechanism by which PriA helicase is bound and stimulated by SSB in Escherichia coli (Ec) has been established, but information on this process in Gram-positive bacteria are limited. We characterized the properties of SSB from Staphylococcus aureus (SaSsbA, a counterpart of EcSSB) and analyzed its interaction with SaPriA. The gel filtration chromatography analysis of purified SaSsbA showed a stable tetramer in solution. The crystal structure of SaSsbA determined at 1.82 Å resolution (PDB entry 5XGT) reveals that the classic oligonucleotide/oligosaccharide-binding folds are formed in the N-terminal DNA-binding domain, but the entire C-terminal domain is disordered. Unlike EcSSB, which can stimulate EcPriA via a physical interaction between EcPriA and the C-terminus of EcSSB (SSB-Ct), SaSsbA does not affect the activity of SaPriA. We also found that SaPriA can be bound by SaSsbA, but not by SaSsbA-Ct. Although no effect was found with SaSsbA, SaPriA can be significantly stimulated by the Gram-negative Klebsiella pneumoniae SSB (KpSSB). In addition, we found that the conserved SSB-Ct binding site of KpPriA (Trp82, Tyr86, Lys370, Arg697, and Gln701) is not present in SaPriA. Arg697 in KpPriA is known to play a critical role in altering the SSB35/SSB65 distribution, but this corresponding residue in SaPriA is Glu767 instead, which has an opposite charge to Arg. SaPriA E767R mutant was constructed and analyzed; however, it still cannot be stimulated by SaSsbA. Finally, we found that the conserved MDFDDDIPF motif in the Gram-negative bacterial SSB is DISDDDLPF in SaSsbA, i.e., F172 in EcSSB and F168 in KpSSB is S161 in SaSsbA, not F. When acting with SaSsbA S161F mutant, the activity of SaPriA was dramatically enhanced elevenfold. Overall, the conserved binding sites, both in EcPriA and EcSSB, are not present in SaPriA and SaSsbA, thereby no stimulation occurs. Our observations through structure-sequence comparison and mutational analyses indicate that the case of EcPriA-EcSSB is not applicable to SaPriA-SaSsbA because of inherent differences among the species.
单链DNA结合蛋白（SSB）和PriA解旋酶在细菌DNA复制重启过程中发挥着重要作用。大肠杆菌（Ec）中PriA解旋酶被SSB结合并激活的机制已经明确，但革兰氏阳性菌中这一过程的相关信息有限。我们对金黄色葡萄球菌的SSB（SaSsbA，EcSSB的对应物）的特性进行了表征，并分析了它与SaPriA的相互作用。对纯化的SaSsbA进行的凝胶过滤色谱分析表明其在溶液中形成稳定的四聚体。以1.82 Å分辨率测定的SaSsbA晶体结构（PDB编号5XGT）显示，经典的寡核苷酸/寡糖结合折叠在N端DNA结合结构域中形成，但整个C端结构域是无序的。与EcSSB不同，EcSSB可通过EcPriA与EcSSB的C末端（SSB-Ct）之间的物理相互作用刺激EcPriA，而SaSsbA不影响SaPriA的活性。我们还发现SaPriA可被SaSsbA结合，但不能被SaSsbA-Ct结合。虽然未发现SaSsbA对其有影响，但革兰氏阴性肺炎克雷伯菌的SSB（KpSSB）可显著刺激SaPriA。此外，我们发现KpPriA的保守SSB-Ct结合位点（Trp82、Tyr86、Lys370、Arg697和Gln701）在SaPriA中不存在。已知KpPriA中的Arg697在改变SSB35/SSB65分布中起关键作用，但SaPriA中的相应残基是Glu767，其电荷与Arg相反。构建并分析了SaPriA E767R突变体；然而，它仍然不能被SaSsbA刺激。最后，我们发现革兰氏阴性菌SSB中保守的MDFDDDIPF基序在SaSsbA中为DISDDDLPF，即EcSSB中的F172和KpSSB中的F168在SaSsbA中为S161，而非F。当与SaSsbA S161F突变体共同作用时，SaPriA的活性显著增强了11倍。总体而言，EcPriA和EcSSB中的保守结合位点在SaPriA和SaSsbA中不存在，因此不会发生刺激作用。我们通过结构-序列比较和突变分析的观察结果表明，由于物种间的固有差异，EcPriA-EcSSB的情况不适用于SaPriA-SaSsbA。
