Protonation and conformational states of a catalytically important histidine sidechain at constant pH

Abstract

We present a benchmarking study to determine the optimal frequency, m of Monte Carlo (MC) steps to attempt changes in the protonation states of titratable sidechains of amino acid residues in constant pH molecular dynamics (CpHMD) simulations. The enzyme, human carbonic anhydrase (HCA) II, having a total of 72 titratable amino acid sidechains, has been used as the template. Along the CpHMD trajectories with \(200 \le m \le 500\) at pH 7.8, HCA II in water is found to exhibit stable structures with equilibrium fluctuations attributed mainly to the loops and turns exposed to the solvent. While the MC sampling step of CpHMD simulations involved all the titratable sites, the present work focuses primarily on the sidechain of catalytically important His-64 due to its well-known role in proton transfer in terms of two major sidechain orientations. The distribution of water molecules around the outward sidechain conformation of His-64 is found to be more sensitive to the choice of m than that around the Zn(II) ion deep inside the active site cavity. The underlying free energy and dynamics of the His-64 sidechain also showed a strong dependence on m. In both cases, an optimal convergence is observed for \(m = 400\)–500 where the fraction of protonated and neutral tautomers of His-64 sidechain accurately reproduce the results derived from a long-timescale CpH-REMD simulation and experimentally determined values of \(pK_a\).

Graphical abstract

synopsis An optimal frequency, m of 400-500 of Monte Carlo steps is reported to attempt changes in protonation states of titratable amino acid sidechains in constant-pH MD simulations by comparing against NMR results. The choice of m on stable conformations of His-64 sidechain of human carbonic anhydrase II is also highlighted.