Initial coordinates of active-state β2R bound to agonist BI-167107 were downloaded from PDB entry 4LDE (30). The BI-167107–bound β2R crystal structure was determined using a β2R-T4 lysozyme (β2R-T4L) fusion protein in which the T4L was fused to the N terminus of the receptor in the presence of camelid antibody fragment. We omitted T4L and camelid antibody fragment from all of our MD simulations. In addition, unresolved parts of IL3, N terminus, and C terminus were omitted from the simulations. Four mutations (M96T, M98T, N187E, and C265A according to UNIPROT numbering) that were introduced in the β2R crystal structure were mutated back to WT residues. Missing atoms of residues Lys60, Glu62, Lys149, Phe223, Gln224, Gln231, Lys263, Phe264, and Lys270 were added using Maestro (Schrödinger, LLC). Asp792.50, Glu1223.41, and Asp1303.49 were protonated as described previously (70).

Prepared receptor-ligand complexes were inserted into explicit palmitoyl-2-oleoylphosphatidylcholine (POPC) lipid bilayer environment using the Desmond MD System (version 4.5; D.E. Shaw Research, New York, NY). The system charges were neutralized, and 150 mM NaCl was added. Overall, the simulation systems consisted of ~107,889 atoms containing 297 lipid molecules, 58 sodium ions, 67 chloride ions, and 21,092 explicit water molecules. To elucidate how Ala substitution for Tyr5.38, which lies toward the extracellular side in β2R, affects β-arrestin interactions with the intracellular side of the receptor, in silico β2R-Y1995.38A MD simulation systems were prepared from representative frames from equilibrated β2R-WT MD simulation trajectories.

MD simulation systems were simulated using Desmond MD System (version 4.5; D.E. Shaw Research, New York, NY) with the OPLS3 force field (71) and TIP3P water model. The protein-membrane relaxation was carried out with a protocol modified from that developed by Schrödinger, LLC. Briefly, the MD simulations were energy minimized and equilibrated for 1 ns with restraints on all protein and ligand-heavy atoms and then were equilibrated for 12 ns with restraints only on the protein backbone and ligand-heavy atoms. For both the equilibrations and the following unrestrained production runs, we used Langevin constant pressure and temperature dynamical system (72) to maintain the pressure at 1 atm and the temperature at 310 K, on an anisotropic flexible periodic cell with a constant-ratio constraint applied on the lipid bilayer in the X-Y plane. For both β2R-WT and β2R-Y1995.38A, we collected 14 trajectories with an aggregated simulation length of 21.0 μs (table S7).

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