MD simulations of the βarr2-APH1A complex (WT and mutated)

All atom MD systems with explicit membrane were set-up using GHARMM-GUI membrane builder (Jo et al., 2009), and simulations were performed using NAMD (Phillips et al., 2005) with the CHARMM36m force field (Huang et al., 2017a) for proteins and the CHARMM36 lipids (Klauda et al., 2010), and the TIP3P water model. We performed two independent runs on each model of the complex explained above, namely Alignment, DOCK1 and DOCK2. Typically, a given system comprised approximately 675 POPC lipid molecules, 78,080 water molecules, 215 Na⁺ ions, and 228 Cl⁻ ions, i.e. a total of 340,560 atoms in a 160 Å × 160 Å × 142 Å box. We relaxed the systems using the equilibration steps in CHARMM-GUI and performed NPT dynamics for 40 ns with 2 fs time step. Nosé-Hoover constant pressure (1 bar) and temperature (310 K) were used. To study the effect of mutations on the complex, we generated three additional runs each containing one mutation (L72E and S75R in βarr2, and R184D in APH1A), for DOCK1 model and the same protocol as that described above. For trajectory analysis and visualization we used VMD (Humphrey et al., 1996) and Pymol (version 1.8).