Implementation and Validation of Titratable Cysteine in GROMACS-Based Constant-pH Molecular Dynamics

Cysteine is a chemically versatile amino acid whose protonation state is central to catalysis, redox regulation, and covalent ligand recognition. Despite the growing availability of constant-pH molecular dynamics (CpHMD) methods, cysteine is not included among the standard titratable residues of the GROMACS-based λ-dynamics implementation developed by Aho, Buslaev, and co-workers. Here, we introduce a titratable cysteine residue, CYST, for this framework and integrate it into the phbuilder workflow. The implementation describes the thiol/thiolate equilibrium of reduced cysteines within a fixed redox topology, while disulfide-linked cysteines are retained as nontitratable covalent states defined a priori. The new residue was calibrated on an ALA-CYS-ALA (ACA) tripeptide by refining the correction potential required to remove the intrinsic force-field bias along the λ coordinate. The resulting model reproduces the expected sigmoidal titration behavior of the peptide and yields a pKa of 8.33, matching the target value adopted in the calibration procedure. Transferability to proteins was then assessed using two complementary benchmarks. First, six engineered single-cysteine mutants of acyl-coenzyme A binding protein (ACBP) were used as a set of noncatalytic cysteines spanning different local environments. The calculations reproduce the overall high-pKa regime of these residues, while also showing that quantitative accuracy depends sensitively on the local conformational ensemble sampled around the introduced cysteine. Second, the implementation was applied to the reactive Cys106 of DJ-1, placing its titration in the correct acidic regime. Overall, this work establishes a practical cysteine extension of the GROMACS CpHMD framework for biomolecular simulations.