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Free-energy profiles for the activation/deactivation of the β 2 -adrenergic receptor (ADRB2) with neutral antagonist and inverse agonist ligands have been determined with well-tempered multiple-walker (MW) metadynamics simulations. The inverse agonists carazolol and ICI118551 clearly favor single inactive conformational minima in both the binary and ternary ligand-receptor-G-protein complexes, in accord with the inverse-agonist activity of the ligands. The behavior of neutral antagonists is more complex, as they seem also to affect the recruitment of the G-protein. The results are analyzed in terms of the conformational states of the well-known microswitches that have been proposed as indicators of receptor activity.

We use enhanced-sampling simulations with an effective collective variable to study the activation of the β 2 -adrenergic receptor in the presence of ligands with different efficacy. The free-energy profiles are computed for the ligand-free ( apo ) receptor and binary ( apo -receptor + G-protein α-subunit and receptor + ligand) and ternary complexes. The results are not only compatible with available experiments but also allow unprecedented structural insight into the nature of GPCR conformations along the activation pathway and their role in the activation mechanism. In particular, the simulations reveal an unexpected mode of action of partial agonists such as salmeterol and salbutamol that arises already in the binary complex without the G-protein. Specific differences in the polar interactions with residues in TM5, which are required to stabilize an optimal TM6 conformation that facilitates G-protein binding and receptor activation, play a major role in differentiating them from full agonists.