Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts.
Department of Chemistry, University of North Texas, Denton, Texas.
Biophys J. 2019 Aug 6;117(3):587-601. doi: 10.1016/j.bpj.2019.06.035. Epub 2019 Jul 5.
The ring-shaped sliding clamp proteins have crucial roles in the regulation of DNA replication, recombination, and repair in all organisms. We previously showed that the Escherichia coli β-clamp is dynamic in solution, transiently visiting conformational states in which Domain 1 at the dimer interface is more flexible and prone to unfolding. This work aims to understand how the stability of the dimer interface influences clamp-opening dynamics and clamp loading by designing and characterizing stabilizing and destabilizing mutations in the clamp. The variants with stabilizing mutations conferred similar or increased thermostability and had similar quaternary structure as compared to the wild type. These variants stimulated the ATPase function of the clamp loader, complemented cell growth of a temperature-sensitive strain, and were successfully loaded onto a DNA substrate. The L82D and L82E I272A variants with purported destabilizing mutations had decreased thermostability, did not complement the growth of a temperature-sensitive strain, and had weakened dimerization as determined by native trapped ion mobility spectrometry-mass spectrometry. The β L82E variant had a reduced melting temperature but dimerized and complemented growth of a temperature-sensitive strain. All three clamps with destabilizing mutations had perturbed loading on DNA. Molecular dynamics simulations indicate altered hydrogen-bonding patterns at the dimer interface, and cross-correlation analysis showed the largest perturbations in the destabilized variants, consistent with the observed change in the conformations and functions of these clamps.
环形滑动夹蛋白在所有生物中对 DNA 复制、重组和修复的调节起着至关重要的作用。我们之前曾表明，大肠杆菌 β 夹在溶液中是动态的，它会短暂地进入构象状态，在这种状态下，二聚体界面上的结构域 1 更灵活，更容易展开。这项工作旨在通过设计和鉴定夹上的稳定和不稳定突变，了解二聚体界面的稳定性如何影响夹的开口动力学和加载。与野生型相比，具有稳定突变的变体赋予了相似或更高的热稳定性，并且具有相似的四级结构。这些变体刺激了夹加载器的 ATP 酶功能，补充了温度敏感菌株的生长，并成功加载到 DNA 底物上。据推测具有不稳定突变的 L82D 和 L82E I272A 变体，其热稳定性降低，不能补充温度敏感菌株的生长，并且通过天然捕获离子迁移率谱-质谱法确定其二聚化减弱。β L82E 变体的熔点降低，但仍能二聚化并补充温度敏感菌株的生长。所有三个具有不稳定突变的夹都对 DNA 的加载产生了干扰。分子动力学模拟表明二聚体界面上氢键模式发生改变，互相关分析表明在不稳定变体中存在最大的扰动，这与这些夹的构象和功能的观察到的变化一致。
