Exploring biased activation characteristics by molecular dynamics simulation and machine learning for the μ-opioid receptor

. The G protein-biased agonist induces greater outward movements of the TM6 intracellular end, forming a typical active conformation, while the β-arrestin-biased agonist leads to a smaller extent of outward movements of TM6. Compared with TRV130, endomorphin2 causes more pronounced inward movements of the TM7 intracellular end and more complex conformational changes of H8 and ICL1. In addition, important residues determining the two different biased activation states were further identified by using an interpretable deep learning classification model, including some common biased activation residues across Class A GPCRs like some key residues on the TM2 extracellular end, ECL2, TM5 intracellular end, TM6 intracellular end, and TM7 intracellular end, and some specific important residues of ICL3 for μOR. The observations will provide valuable information for understanding the biased activation mechanism for GPCRs.