Department of Physics, State University of Ponta Grossa (UEPG), Ponta Grossa,Paraná, Brazil.
Department of Chemistry, State University of Ponta Grossa (UEPG), Ponta Grossa, Paraná, Brazil.
PLoS One. 2019 Apr 18;14(4):e0214601. doi: 10.1371/journal.pone.0214601. eCollection 2019.
RecA is a multifunctional protein that plays a central role in DNA repair in bacteria. The structural Make ATP Work motif (MAW) is proposed to control the ATPase activity of RecA. In the present work, we report the biochemical activity and structural effects of the L53Q mutation at the MAW motif of the RecA protein from H. seropedicae (HsRecA L53Q). In vitro studies showed that HsRecA L53Q can bind ADP, ATP, and ssDNA, as does wild-type RecA. However, the ATPase and DNA-strand exchange activities were completely lost. In vivo studies showed that the expression of HsRecA L53Q in E. coli recA1 does not change its phenotype when cells were challenged with MMS and UV. Molecular dynamics simulations showed the L53Q point mutation did not cause large conformational changes in the HsRecA structure. However, there is a difference on dynamical cross-correlation movements of the residues involved in contacts within the ATP binding site and regions that hold the DNA binding sites. Additionally, a new hydrogen bond, formed between Q53 and T49, was hypothesized to allow an independent motion of the MAW motif from the hydrophobic core, what could explain the observed loss of activity of HsRecA L53Q.
RecA 是一种多功能蛋白，在细菌的 DNA 修复中起着核心作用。结构上的 ATP 作用基序（MAW）被认为可以控制 RecA 的 ATP 酶活性。在本工作中，我们报告了来自 H. seropedicae（HsRecA L53Q）的 RecA 蛋白 MAW 结构域中 L53Q 突变的生化活性和结构影响。体外研究表明，HsRecA L53Q 可以像野生型 RecA 一样结合 ADP、ATP 和 ssDNA。然而，ATP 酶和 DNA 链交换活性完全丧失。体内研究表明，在大肠杆菌 recA1 中表达 HsRecA L53Q 时，当细胞受到 MMS 和 UV 挑战时，其表型不会改变。分子动力学模拟表明，L53Q 点突变不会导致 HsRecA 结构的大构象变化。然而，在涉及 ATP 结合位点内和 DNA 结合位点的残基之间的接触的动态交叉相关运动方面存在差异。此外，假设形成了 Q53 和 T49 之间的新氢键，允许 MAW 基序从疏水区独立运动，这可以解释观察到的 HsRecA L53Q 活性丧失。
