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

fi2AR is an important drug target protein involving many diseases. Biased drugs induce specific signaling and provide additional clinical utility to optimize fi2AR-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 fi-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 fi- arrestin-biased agonists regulate an occluded conformation with a slightly outward movement of TM6 and an inward shift of TM7, which should favor fi-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 fi-arrestin-biased agonists induce ICL3 to form a closed conformation with a stable local a-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 fi-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 fi-arrestin-biased agonists forms additional hydrophobic interactions with W6.48 and F6.51 after the rearrangement, which should contribute to the fi-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 fi-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 fi-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.

Automated summary

This study explores the molecular mechanisms of biased activation induced by structural differences between different agonists. It reveals that G-protein-biased agonists and fi-arrestin-biased agonists achieve biased signaling through different conformational changes. In addition, the study also identifies specific conformations of ICL3 under specific agonists and the roles of key residues. These findings contribute to a better understanding of biased activation mechanisms and provide guidance for the design of biased drugs.