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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.

In this work, we combined accelerated molecular dynamics (aMD) and conventional molecular dynamics (cMD) simulations coupled with the potential of mean force (PMF), correlation analysis, principal component analysis (PCA), and protein structure network (PSN) to study the effects of dimerization and the mutations of I52V and V150A on the CCR5 homodimer, in order to elucidate the mechanism regarding cooperativity of the ligand binding between two protomers and to address the controversy about the mutation-induced dimer-separation. The results reveal that the dimer with interface involved in TM1, TM2, TM3, and TM4 is stable for the CCR5 homodimer. The dimerization induces an asymmetric impact on the overall structure and the ligand-binding pocket. As a result, the two protomers exhibit an asymmetric binding to the maraviroc (one anti-HIV drug). The binding of one protomer to the drug is enhanced while the other is weakened. The PSN result further reveals the allosteric pathway of the ligand-binding pocket between the two protomers. Six important residues in the pathway were identified, including two residues unreported. The results from PMF, PCA, and the correlation analysis clearly indicate that the two mutations induce strong anticorrelation motions in the interface, finally leading to its separation. The observations from the work could advance our understanding of the structure of the G protein-coupled receptor dimers and implications for their functions.

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