Chloride Binding in Trimeric Coiled Coils: Free Energy and Structural Determinants from Molecular Simulations

Coiled coils, owing to their simple yet versatile architecture, serve as valuable model systems for both experimental and computational studies in protein science. Whereas the sequence-structure relationships that govern their oligomeric state and stability have been thoroughly investigated, important gaps remain, most notably regarding the role of central chloride ions coordinated by asparagine triads observed in several trimeric coiled-coil (TCC) crystal structures. To investigate the thermodynamics of chloride binding at this site, we performed extensive molecular simulations using metadynamics and alchemical free-energy calculations, both enhanced with replica exchange, to determine the chloride binding free energy (ΔG_bind) in three TCCs of similar length but different stability (PDB IDs: 2wpy, 4dzk, 1mof). Despite the nearly identical local coordination environment, the computed ΔG_bind values strongly depend on the overall protein structure, with variations in superhelical radius R₀ upon ion removal systematically accompanying the observed binding thermodynamics. In particular, both the metastable TCC 2wpy─a variant of the GCN4 leucine-zipper domain previously shown to be unstable in the absence of chloride─and the synthetic design 4dzk exhibit highly unfavorable binding, suggesting that current biomolecular force fields may not fully capture either the stabilizing role of chloride or the conformational ensemble of the unbound state. By contrast, the calculated ΔG_bind in 1mof, a fragment of the MoMuLV retroviral transmembrane protein, is favorable and is associated with the presence of an additional C-terminal leash domain that modulates the binding-site environment. These results identify TCCs as critical benchmarks for improving the description of anion-protein interactions and the balance between bound and unbound states in future force-field developments.