Molecular Mechanisms of Diverse Activation Stimulated by Different Biased Agonists for the β2-Adrenergic Receptor

β2AR is an important drug target protein involving many diseases. Biased drugs induce specific signaling and provide additional clinical utility to optimize β2AR-based therapies. However, the biased signaling mechanism has not been elucidated. Motivated by the issue, we chose four agonists with divergent bias (balanced agonist, G-protein-biased agonist, and β-arrestin-biased agonists) and utilized Gaussian accelerated molecular dynamics simulation coupled with a dynamic network to probe the molecular mechanisms of distinct biased activation induced by the structural differences between the four agonists. Our simulations reveal that the G-protein-biased agonist induces an open conformation with the outward shifts of TM6 and TM7 for the intracellular domain, which will be beneficial to couple G protein. In contrast, the β-arrestin-biased agonists regulate an occluded conformation with a slightly outward movement of TM6 and an inward shift of TM7, which should favor β-arrestin signaling. The balanced agonist does not induce an observable outward shift for TM6 but, along with a slight tilt for TM7, leads to an inactive-like conformation. In addition, our results reveal the first time that ICL3 presents specific conformations with different agonists. The G-protein-biased agonist drives ICL3 to open so that the G protein-binding pocket can be available, while the β-arrestin-biased agonists induce ICL3 to form a closed conformation with a stable local α-helix. MM/PBSA analysis further reveals that the hydroxyl groups in the resorcinol of the G-protein-biased agonist form strong interactions with Y5.38 and S5.42, thus preventing tilting of the TM5 extracellular end. The catechol of the balanced agonist and the β-arrestin-biased ones induces the rearrangement of two hydrophobic residues F6.52 and W6.48. However, different from the balanced agonist, the ethyl substituent of β-arrestin-biased agonists forms additional hydrophobic interactions with W6.48 and F6.51 after the rearrangement, which should contribute to the β-arrestin bias. The shortest pathway analysis further reveals that the three residues Y7.43, N7.45, and N7.49 are crucial for allosterically regulating G-protein-biased signaling, while the two residues W6.48 and F6.44 make an important contribution to regulate β-arrestin-biased signaling. For the balanced agonist NE, the allosteric regulation pathway simultaneously involves the residue associated with G-protein-biased signaling like S5.46 and the residues related to β-arrestin-biased signaling like W6.48 and F6.44, thus producing unbiased signaling. The observations could advance our understanding of the biased activation mechanism on class A GPCRs and provide a useful guideline for the design of biased drugs.