Constant-pH simulation of the humanβ₂adrenergic receptor inactivation

Understanding the molecular basis of pH-dependent G protein-coupled receptor (GPCR) signaling is crucial for comprehending physiological regulation and drug design. Here, we investigate the humanβ₂adrenergic receptor (β₂AR), a prototypical GPCR whose function is sensitive to pH conditions. Employing extensive constant-pH molecular dynamics simulations, we provide a detailed atomistic characterization ofβ₂AR inactivation across physiologically relevant pH values (4–9). Our simulations reveal thatβ₂AR inactivation is closely linked to protonation events at critical residues, notably E268^6×30involved in the ionic lock formation. Furthermore, we find that inactivation occurs without direct sodium binding to the ion-binding pocket around residue D79^2×50. Instead, sodium ions predominantly interact with D113^3×32, effectively blocking deeper entry toward the traditional binding site. These results challenge existing mechanistic models and highlight the necessity of accurately modeling electrostatics in GPCR simulations. Our findings underscore the potential of constant-pH methodologies to advance the understanding of GPCR dynamics, influencing both fundamental biology and therapeutic strategies.