Molecular Dynamics Simulations of Receptor and Regulatory Complexes

MD simulations of Rec and NCS1 states were performed using GROMACS 2016.1 simulation package (Abraham et al., 2015 (link)) and CHARMM36m (Huang et al., 2017 (link)) all-atom force field, where parameters for N-myristoylated Gly were generated manually (available upon request). Simulations were performed in a dodecahedral box with Periodic Boundary Conditions applied, where proteins were located at 12 Å distance from box boundaries. Solvent was modeled as TIP3P water, system was neutralized first with 1 mM MgCl₂, then after addition of 150 mM KCl. The size for each simulated system was the following: 33,837 atoms for Rec-T, 38,878 atoms for Rec-I, 56,010 atoms for Rec-R, 86,456 atoms for Rec-GRK1, 46,809 atoms for NCS1-iso, 42,882 atoms for NCS1- D₂R and 50,584 atoms for NCS1-GRK1.
All structures underwent substantially the same pre-production steps as previous studies concerning GCAP1 (Marino et al., 2015b (link)), briefly consisting of steepest descent (F = 1,000 kJ/mol^*nm) and conjugate gradients (F = 500 kJ/mol^*nm) energy minimization, then equilibration at 310 K for 2 ns backbone-constrained and 2 ns unrestricted MD simulations in NVT ensemble. After equilibration, 200 ns unrestrained MD simulations were performed in NPT ensemble at 310 K and 1 atm for each system. Equilibration and production MD simulations were independently replicated 5 times by changing the random seed for initial velocity generation as previously described (Marino and Dell'orco, 2016 (link)), to achieve exhaustive sampling of the conformational space.