Structural Insights into Allosteric Modulation of GPCRs

A total of 18 unique experimental structures and 8 computational models of allosteric modulator-bound class A and B GPCRs were prepared for simulations (Fig. 1A and Supplementary Table 1). The GPCR structures bound by NAMs included the MT7-bound M₁R (PDB: 6WJC)^{19 (link)}, Cmpd-15-bound β₂AR (PDB: 5X7D)^{27 (link)}, AS408-bound β₂AR (PDB: 6OBA)^{29 (link)}, NDT9513727-bound C5AR1 (PDB: 6C1Q)³⁰ , Avacopan-bound C5AR1 (PDB: 6C1R)³⁰ , ORG27569-bound CB₁ receptor (PDB: 6KQI)^{31 (link)}, GTPL9431-bound CCR2 (PDB: 5T1A)^{32 (link)}, NNC0640-bound GLP1R (PDB: 5VEX)^{35 (link)}, PF-06372222-bound GLP1R (PDB: 6LN2)³⁶ , and MK-0893-bound GLR (PDB: 5EE7)^{23 (link)}. Six computational models of NAM-bound GPCRs included the MT7-bound M₂R and M₄R, which were built by aligning the 6WJC PDB structure of M₁R to the 5ZK3^{57 (link)} and 5DSG^{58 (link)} PDB structures of M₂R and M₄R, respectively, and copying atomic coordinates of the atropine antagonist and MT7 NAM, as well as the Cmpd-15-bound α_1B-adrenoceptor (α_1BAR), α_2A-adrenoceptor (α_2AAR), α_2C-adrenoceptor (α_2CAR), and β₁-adrenoceptor (β₁AR), which were built by aligning the 5X7D PDB structure of β₂AR to the 7B6W⁵⁹ , 6KUX⁶⁰ , 6KUW⁶¹ , and 7BVQ^{62 (link)} PDB structures of α_1BAR, α_2AAR, α_2CAR, and β₁AR, respectively, and copying atomic coordinates of the carazolol antagonist and Cmpd-15 NAM. The GPCR structures bound by PAMs included the MIPS521-bound A₁AR (PDB: 7LD3)^{24 (link)}, LY2119620-bound M₂R (PDB: 6OIK)^{17 (link)}, LY2119620-bound M₄R (PDB: 7V68)²⁵ , Cmpd-6FA-bound β₂AR (PDB: 6N48)^{28 (link)}, LY3154207-bound D₁R (PDB: 7LJC)^{33 (link)}, AgoPAM-bound FFAR1 (PDB: 5TZY)²⁰ , INT777-bound GPBAR (PDB: 7CFN)^{34 (link)}, and LSN3160440-bound GLP1R (PDB: 6VCB)^{22 (link)}. Two computational models of PAM-bound GPCRs included LY2119620-bound M₁R, which was built by aligning the 6OIK PDB structure of M₂R to the 6OIJ PDB structure of M₁R^{17 (link)} and copying atomic coordinates of the LY2119620 PAM, and LY32154207-bound D₂ receptor (D₂R), which was built by aligning the 7LJC PDB structure of D₁R to the 7JVR PDB structure of D₂R^{63 (link)} and copying atomic coordinates of the SKF-81297 agonist and LY3154207 PAM.
SWISS-MODEL^{64 (link)} homology modeling was applied to restore missing residues in the GPCR structures and models, particularly in the ECL2, ICL2, and ECL3. Charges of the ligands were listed in Supplementary Table 1. All water and heteroatom molecules except the ligands and receptor-bound ions (including the sodium ion in the 6C1R PDB structure of C5AR1 and zinc ion in the 6LN2 PDB structure of GLP-1 receptor) were removed from the structures. The GPCR complexes were embedded in POPC membrane lipid bilayers and solvated in 0.15M NaCl (Fig. 1B). The AMBER^{65 (link)} force field parameter sets were used for our GaMD simulations, specifically ff19SB^{66 (link)} for proteins, GAFF2^{67 (link)} for ligands, LIPID17 for lipids, and TIP3P⁶⁸ for water, except for the A₁AR simulations as obtained from a previous study^{24 (link)} where the CHARMM36m^{69 (link)} force field parameter set was used.