Seol Yeonee, Tse-Dinh Yuk-Ching, Neuman Keir C
Laboratory of Single Molecule Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
Department of Chemistry & Biochemistry, Florida International University, Miami, FL 33199, USA.
Res Sq. 2025 Jul 14:rs.3.rs-7087376. doi: 10.21203/rs.3.rs-7087376/v1.
Protein conformational dynamics are fundamental to enzyme function, yet the molecular mechanisms by which these dynamics are regulated remain poorly understood. Here, we reveal that a conserved network of salt-bridges, modulated by magnesium ions, serves as a key regulator of conformational transitions in Type IA topoisomerases (TopIA). Using a combination of molecular dynamics simulations, targeted protein mutagenesis, and functional assays, we demonstrate that Mg binding to a previously unrecognized metal binding site orchestrates the opening and closing of the protein-mediated DNA gate-a critical step in TopIA's catalytic cycle. Our results show that magnesium tunes the kinetics of the salt-bridge network's configurational switching, directly impacting enzyme activity and providing a safeguard against DNA damage under Mg depletion. This work provides a new chemical and structural framework for understanding divalent cation-dependent regulation of protein function via networked salt-bridges. Our findings open new avenues for the rational design of cation-sensitive proteins and inhibitors, and highlight an evolutionarily conserved strategy for coupling environmental sensing to molecular function.
蛋白质构象动力学是酶功能的基础，然而这些动力学的调节分子机制仍知之甚少。在这里，我们揭示了一个由镁离子调节的保守盐桥网络，它是IA型拓扑异构酶（TopIA）构象转变的关键调节因子。通过结合分子动力学模拟、靶向蛋白质诱变和功能测定，我们证明镁与一个以前未被识别的金属结合位点结合，协调了蛋白质介导的DNA门的打开和关闭——这是TopIA催化循环中的关键一步。我们的结果表明，镁调节盐桥网络构型转换的动力学，直接影响酶活性，并在镁缺乏时提供防止DNA损伤的保护机制。这项工作为理解通过网络化盐桥对蛋白质功能进行二价阳离子依赖性调节提供了一个新的化学和结构框架。我们的发现为阳离子敏感蛋白质和抑制剂的合理设计开辟了新途径，并突出了一种将环境感知与分子功能相耦合的进化保守策略。
