Protonation-Coupled Rewiring of the Hydrogen-Bond Network Underlies Allosteric Dysfunction in the ALS-Linked K76E Mutant of Cyclophilin A

Abstract The recently discovered K76E mutation in cyclophilin A (PPIA) is associated with amyotrophic lateral sclerosis (ALS), yet the atomic-level mechanism driving its dysfunction remains elusive. Using constant pH molecular dynamics (CpHMD) simulations, we find that the distal K76E substitution is accompanied by changes in the protonation states of key histidine residues and in their local conformational environment. Because these protonation populations derive from a single 100 ns CpHMD trajectory per system, they may be sensitive to limited conformational sampling, and we treat them as provisional assignments rather than as converged equilibrium properties. In microsecond-scale fixed-charge simulations with these assignments, the hydrogen-bonding network around the active site is reorganized and the occupancy of contacts near the catalytic site is altered. The catalytic gatekeeper loops become more flexible and the correlation-based communication pathway linking the mutation site to the catalytic center is altered, without large-scale structural change. Our results suggest that coupled protonation–conformational changes, rather than direct active-site disruption, may contribute to the reduced activity of the K76E variant, underscoring the often-overlooked role of variable protonation states in disease-associated mutations; establishing this mechanism will require more extensive CpHMD sampling.