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 distinct 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.
