pH-Driven β₂AR Dynamics Reveal Loop-Mediated Allosteric Communication.

Area of Science:

• Biochemistry
• Computational Biology
• Structural Biology

Background:

• Membrane protein structure and dynamics are sensitive to environmental factors like pH.
• Changes in pH alter the protonation states of ionizable residues, affecting protein electrostatics and stability.
• Constant-pH molecular dynamics (CpHMD) allows dynamic proton exchange, simulating pH effects on proteins.

Purpose of the Study:

• To investigate the influence of pH variations on the local conformational behaviors of the beta-2 adrenergic receptor (β2AR).
• To explore how dynamic protonation changes affect receptor loop flexibility and communication pathways.

Main Methods:

• Applied constant-pH molecular dynamics (CpHMD) at pH 6.5, 7.0, and 8.0.
• Performed conventional molecular dynamics (MD) with fixed protonation states for comparison.
• Analyzed loop flexibility, hydrogen-bonding patterns, and communication between receptor regions using mutual information analysis.

Main Results:

• Loop regions rich in titratable residues (ICL3, ECL2) showed the most significant response to pH changes.
• CpHMD revealed pH-dependent redistribution of loop flexibility and hydrogen-bonding, unlike constrained behavior in fixed-protonation simulations.
• Key GPCR microswitches remained in inactive states, indicating pH primarily affects local loop dynamics.

Conclusions:

• pH variations modulate the local dynamics and flexibility of the beta-2 adrenergic receptor (β2AR) through protonation state changes.
• Altered communication between extracellular and intracellular loops was observed under different pH conditions.
• Incorporating pH effects in molecular simulations is crucial for understanding GPCR behavior.