pH-Driven β2AR Dynamics Reveal Loop-Mediated Allosteric Communication

Abstract

Membrane protein structure and dynamics are highly sensitive to environmental conditions, including changes in pH that can alter the protonation states of ionizable residues and, in turn, influence local electrostatics and stability. Constant-pH molecular dynamics (CpHMD) provides a framework to explore such effects by allowing dynamic proton exchange during simulations. Here, we applied CpHMD at pH:6.5, 7.0, and 8.0, alongside conventional MD, to examine how pH variations may influence the local conformational behaviors of the beta 2-adrenergic receptor (beta 2AR). During the 1.2-mu s-long total simulation, loop regions rich in titratable residues, particularly ICL3 and ECL2, showed the strongest responses to protonation changes. CpHMD trajectories suggested a pH-dependent redistribution of loop flexibility and hydrogen-bonding patterns, producing a see-saw-like effect, while fixed-protonation Control runs showed more constrained behavior. Across all simulations, the key GPCR microswitches, such as the ionic lock, the Y-Y gate, the NPxxY and PIF motifs, and the Trp286-Phe290 toggle pair, stayed within the ranges expected for an inactive receptor. This suggests that pH changes mainly influence local loop motions in the inactive receptor without pushing it toward activation-like states. Finally, mutual information analysis on both C alpha atoms and dihedral angles revealed altered communication between the extracellular and intracellular loops under different pH environments. While limited in time scale, these results provide a computational perspective on how protonation dynamics can modulate the GPCR behavior and highlight the value of incorporating pH effects in molecular-level investigations.