Structural Features and Ligand Selectivity for 10 Intermediates in the Activation Process of β

It has already been suggested by researchers that there should be multiple intermediate states in the activation process for G-protein-coupled receptors (GPCRs). However, the intermediate states are very short-lived and hardly captured by the experiments, leading to very limited understanding of their structural features and drug efficacies. In this work, a novel joint strategy of targeted molecular dynamics simulation, conventional molecular dynamics simulation, and virtual screening is developed to address the problems. The results from 10 intermediate conformations obtained from the work reveal that the ligand pocket is very unstable and fluctuates between the inactive state and the active one in the case of ligand-free, in particular for ECL2 as a gate-keeper of the ligand-binding. The ligand-binding site could be stable in the active state with a small volume and a completely closed ECL2, only when the G-protein-binding region is fully activated. In addition, the activations of the ligand-binding pocket and G-protein-binding site are relatively independent and exhibit a loose allosteric coupling, which contributes to the existence of multiple intermediate conformations. Interestingly, the screening performance of the agonists does not increase on increasing the overall activity of the intermediate state, but is dependent on the activated extent of the ligand pocket. The receptor is prone to bind the agonist when closing ECL2 and reducing the ligand-binding pocket volume, whereas it is more favorable for binding the antagonist when opening ECL2 and increasing the pocket volume. These observations added to previous studies could help us better understand the activation mechanism of GPCRs and provide valuable information for drug design.