Simulations of the μ opioid receptor (μOR) were based on both the antagonist-bound inactive-state crystal structure (PDB ID: 4DKL) and the agonist-bound active-state crystal structure described in this manuscript. Coordinates were prepared by first removing all non-ligand and non-receptor molecules except for the cholesterol neighboring TM7 and for crystallographic water molecules near the receptor. For inactive μOR simulations, the T4 lysozyme was removed and acetyl and methylamide capping groups were placed on R263ICL3 and E270ICL3. For active μOR simulations, the nanobody was removed. In both cases, Prime (Schrödinger, Inc.) was used to model missing side-chains, and capping groups were then added to the N- and C- termini of the receptor. Histidine residues were simulated as the neutral Nε tautomer. Other titratable residues were simulated in their dominant protonation state at pH 7 except for D1142.50, which was charged in inactive simulations and neutral in active simulations. A sodium ion was placed adjacent to D1142.50 in inactive simulations.
The μOR was simulated in seven distinct conditions. These include: (1) The unliganded, inactive μOR, prepared by deleting the covalently bound, co-crystallized ligand, β-FNA, and adding a proton in its place to K2335.39; (2) The inactive μOR with the co-crystallized ligand β-FNA; (3) The inactive μOR with agonist β-FOA (which does not bind covalently); (4) The unliganded, active μOR, prepared by deleting the co-crystallized ligand, BU72; (5) The active μOR with the co-crystallized ligand BU72; (6) The active μOR with the co-crystallized ligand BU72, with the N-terminal peptide deleted; (7) The active μOR with the antagonist BU74, with the N-terminal peptide deleted. Simulations of the active μOR without N-terminal peptide were prepared by deleting residues 52 through 64 of the receptor. Simulations with β-FOA were prepared by docking β-FOA to the crystallographic pose of β-FNA. Simulations with BU74 were prepared by docking BU74 to the crystallographic pose of BU72 and rotating the torsion angle of the methylcyclopropyl group to agree with that of β-FNA's methylcyclopropyl group.
We performed three to six simulations per condition (Supplementary Section). Simulations in a given condition were initiated from identical structures, but with initial atom velocities assigned independently and randomly.
It should be noted that in all liganded simulations, including those with β-FNA, β-FOA, BU72, and BU74, the ligand's tertiary amine nitrogen was protonated and therefore the ligand was simulated as a cation. This is necessary for the ligand to form the conserved salt bridge with neighboring D1473.32.
Each of the resulting prepared μOR receptor structures was then aligned to the Orientations of Proteins in Membranes (OPM)52 (link) entry for the inactive μOR using MacPyMOL (Schrödinger). The μOR was modified with disulfide bridges and inserted into a hydrated, equilibrated palmitoyloleoylphosphatidylcholine (POPC) bilayer using the CHARMM-GUI interface53 (link)-56 (link). Sodium and chloride ions were added to neutralize the system, reaching a final concentration of approximately 150 mM. All simulations contained one μOR receptor embedded in a lipid bilayer with 160 POPC molecules.
The μOR was simulated in seven distinct conditions. These include: (1) The unliganded, inactive μOR, prepared by deleting the covalently bound, co-crystallized ligand, β-FNA, and adding a proton in its place to K2335.39; (2) The inactive μOR with the co-crystallized ligand β-FNA; (3) The inactive μOR with agonist β-FOA (which does not bind covalently); (4) The unliganded, active μOR, prepared by deleting the co-crystallized ligand, BU72; (5) The active μOR with the co-crystallized ligand BU72; (6) The active μOR with the co-crystallized ligand BU72, with the N-terminal peptide deleted; (7) The active μOR with the antagonist BU74, with the N-terminal peptide deleted. Simulations of the active μOR without N-terminal peptide were prepared by deleting residues 52 through 64 of the receptor. Simulations with β-FOA were prepared by docking β-FOA to the crystallographic pose of β-FNA. Simulations with BU74 were prepared by docking BU74 to the crystallographic pose of BU72 and rotating the torsion angle of the methylcyclopropyl group to agree with that of β-FNA's methylcyclopropyl group.
We performed three to six simulations per condition (Supplementary Section). Simulations in a given condition were initiated from identical structures, but with initial atom velocities assigned independently and randomly.
It should be noted that in all liganded simulations, including those with β-FNA, β-FOA, BU72, and BU74, the ligand's tertiary amine nitrogen was protonated and therefore the ligand was simulated as a cation. This is necessary for the ligand to form the conserved salt bridge with neighboring D1473.32.
Each of the resulting prepared μOR receptor structures was then aligned to the Orientations of Proteins in Membranes (OPM)52 (link) entry for the inactive μOR using MacPyMOL (Schrödinger). The μOR was modified with disulfide bridges and inserted into a hydrated, equilibrated palmitoyloleoylphosphatidylcholine (POPC) bilayer using the CHARMM-GUI interface53 (link)-56 (link). Sodium and chloride ions were added to neutralize the system, reaching a final concentration of approximately 150 mM. All simulations contained one μOR receptor embedded in a lipid bilayer with 160 POPC molecules.
