Networked Salt-Bridges Mediate Magnesium-Dependent Conformational Dynamics and Functional Regulation in Type IA Topoisomerases

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 single-molecule and ensemble measurements, molecular dynamics simulations, and targeted protein mutagenesis, we demonstrate that Mg²⁺ binding to a previously unrecognized divalent 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 Mg²⁺ depletion. This work provides a chemical and structural framework for understanding divalent cation-dependent regulation of protein function via networked salt-bridges. Our findings open additional avenues for the rational design of cation-sensitive proteins and inhibitors, and highlight an evolutionarily conserved strategy for coupling environmental sensing to molecular function.