Abstract
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.
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Acknowledgments
This work utilized the computational resources of the NIH HPC Biowulf cluster (https://hpc.nih.gov), NHLBI Biophysics core facility and Biochemistry core facility. We would like to thank Marie-Paule Strub for protein expression vector and purification consultation, Yi He for protein expression, Ian Morgan and Xi Yang for assisting with Topoisomerase 3 cleavage, and Fabrizio Marinelli for molecular dynamics simulation discussion. We thank Robert van Waardenburg and Wei Yang for critical reading of the manuscript. Intramural research program of the National Heart, Lung, and Blood Institute of the National Institutes of Health [1ZIAHL001056] (KCN) and National Institutes of General Medical Sciences [R35GM139817] (YT).
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Seol, Y., Tse-Dinh, YC. & Neuman, K.C. Networked salt-bridges mediate magnesium-dependent conformational dynamics and functional regulation in type IA topoisomerases. Nat Commun (2026). https://doi.org/10.1038/s41467-026-72556-9
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DOI: https://doi.org/10.1038/s41467-026-72556-9
