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

## TL;DR

This study uses molecular simulations to explore how chloride ions bind to trimeric coiled coils and how this affects their stability.

## Contribution

The paper introduces a novel analysis of chloride binding thermodynamics in trimeric coiled coils using advanced simulation techniques.

## Key findings

- Chloride binding free energy varies significantly between different trimeric coiled coils despite similar local environments.
- The presence of a C-terminal leash domain in 1mof enhances favorable chloride binding.
- Current force fields may not accurately capture chloride's stabilizing role or the unbound state conformational ensemble.

## Abstract

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 R
0 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.