Dissociation kinetics of G proteins from G protein–coupled receptors and effects of allosteric modulation

G protein-coupled receptors (GPCRs), the largest superfamily of human membrane proteins with >800 members, are primary targets for ~1/3 of all marketed drugs. Recent fluorescence experiments underscored the pivotal role of GPCR-G protein complex lifetime in their coupling efficiency and selectivity. However, these experiments are often expensive, time-consuming, and limited to a small number of GPCR-G protein systems. On the other hand, it is challenging to simulate GPCR-G protein dissociation using molecular dynamics (MD) methods. Here, we have employed Protein-Protein Interaction Gaussian accelerated MD (PPI-GaMD) simulations and experiments to probe the kinetics and pathways of G protein dissociation from GPCRs. For five systems with published experimental kinetic data, PPI-GaMD simulations successfully captured G protein dissociation from the GPCRs, including the adrenergic, adenosine, and muscarinic receptors. The simulations allowed identification of two distinct dissociation pathways and calculation of the G protein dissociation rates, which were in good agreement with experimental data. Additionally, we simulated the effect of positive allosteric modulators (PAMs) of the adenosine A₁ receptor (A₁R) in Gi protein dissociation and supported simulation findings with bioluminescence resonance energy transfer biosensor experiments evaluating G_βγ kinetics following A₁R activation. A₁R PAMs were found to strengthen the agonist-receptor and receptor-G protein interactions and significantly reduce dissociation rates of the Gi protein. In summary, complementary PPI-GaMD simulations and kinetic assays have enabled detailed characterization of the kinetics and pathways of G protein dissociation, a critical event in the GPCR signaling cascade, and the effects of GPCR allosteric modulators.