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 5ZK357 (link) and 5DSG58 (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 α1B-adrenoceptor (α1BAR), α2A-adrenoceptor (α2AAR), α2C-adrenoceptor (α2CAR), and β1-adrenoceptor (β1AR), which were built by aligning the 5X7D PDB structure of β2AR to the 7B6W59 , 6KUX60 , 6KUW61 , and 7BVQ62 (link) PDB structures of α1BAR, α2AAR, α2CAR, 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 M1R17 (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 D2R63 (link) and copying atomic coordinates of the SKF-81297 agonist and LY3154207 PAM.
SWISS-MODEL64 (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 AMBER65 (link) force field parameter sets were used for our GaMD simulations, specifically ff19SB66 (link) for proteins, GAFF267 (link) for ligands, LIPID17 for lipids, and TIP3P68 for water, except for the A1AR simulations as obtained from a previous study24 (link) where the CHARMM36m69 (link) force field parameter set was used.
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