Investigation of salt-bridge interactions underlying salt activation of halophilic methionine sulfoxide reductase A.

INTRODUCTION: Halophilic microorganisms inhabit hypersaline environments where sustained ionic stress challenges protein structure and function, making salt-responsive enzymes important for cellular fitness. Here, we investigated the salt-bridge interactions that support salt-dependent activation of a halophilic methionine sulfoxide reductase A from Halobacterium hubeiense (HhMsrA), a key enzyme involved in repairing oxidized methionine residues. METHODS: Molecular dynamics (MD) simulations, mutagenesis analyses, enzyme activity assays, and circular dichroism (CD) measurements were used to identify key salt-bridge-forming residues and evaluate their contributions to the salt-dependent structural and functional behavior of HhMsrA. RESULTS: MD simulations and mutagenesis analyses identified Arg106 and Lys76 as prominent salt-bridge nodes, and charge-conservative substitutions at these positions largely preserved the high-salt activity profile, whereas non-conservative substitutions (e.g., acidic, hydrophobic, aromatic, or proline replacements) generally reduced activity. Moreover, combined proline substitutions produced a pronounced cumulative effect, leaving the enzyme with only minimal activity even under high-salt conditions. MD analyses further associated these site-specific salt-bridge interactions with the maintenance of the salt-adapted structural state required for HhMsrA activity. CD measurements further showed a strong KCl-dependent helical response in HhMsrA that was attenuated to varying degrees across mutants targeting salt-bridge-forming residues. DISCUSSION: These findings demonstrate that salt-dependent activity of HhMsrA is supported by a small set of salt-bridge nodes that contribute to maintaining a high-salt structural state compatible with HhMsrA catalytic function.