2.2. All-Atom Molecular Dynamics Simulations

Molecular dynamics simulations were performed with the GROMACS 5 package [42,43,44,45,46] using the Amber99SB-ILDN force field for proteins that has been shown to yield an accurate description of many structural and dynamical properties of proteins [47,48,49,50]. Side chain protonation states of titratable amino acids were assigned using a value of pH = 7.4 with the help of the pdb2pqr software [51]. Capping acetyl and methyl-amino groups were added to the N and C termini of both AC5 domains and Gαi to avoid strong artifacts between non-natural termini [23,36]. The four states we study (Gαi_sym + AC5, Gαi_sym + AC5 + ATP, Gαi_tilted + AC5, Gαi_tilted + AC5 + ATP) were each placed in a truncated octahedral box and solvated with TIP3P water molecules [52] to a depth of at least 11 Å. The solute was neutralized with potassium cations, and then K⁺ Cl⁻ ion pairs [53] were added to reach a physiological salt concentration of 0.15 M. Parameters for ATP and GTP were taken from [54]. The parameters for Mg²⁺ came from [55]. This new set of parameters was developed to improve the kinetic properties of Mg²⁺ ions with water and with the phosphate ion, and it was implemented in Amber99. This new set of parameters also provided a better description of the structure of Mg²⁺-phosphate binding than previous sets (these interactions are naturally important in our simulations in the presence of ATP) [55]. Hence, the combination of Amber 99SB-ILDN and the new set of parameters of Mg²⁺ ions is currently the best choice to reproduce the dynamics of AC5 and Gsα, and to properly describe the interactions of Mg²⁺ with AC5 and ATP.

Long-range electrostatic interactions were treated using the particle mesh Ewald method [56,57] with a real-space cutoff of 10 Å. We used virtual interaction sites for the hydrogens, and bond lengths were restrained using P-LINCS [45,58], allowing a time step of 4 fs [59]. The translational movement of the solute was removed every 1000 steps to avoid any kinetic energy build-up [60]. After energy minimization of the solvent and equilibration of the solvated system for 10 ns using a Berendsen thermostat (τ_T = 1 ps) and Berendsen pressure coupling (τ_P = 4 ps) [61], the simulations were carried out in an NTP ensemble at a temperature of 310 K and a pressure of 1 bar using a Bussi velocity-rescaling thermostat [62] (τ_T = 1 ps) and a Parrinello–Rahman barostat (τ_P = 1 ps) [63]. Simulations were carried out using typically between 72 and 120 computer cores depending on the system size, which allowed a production rate of about 100 ns/day. Analysis was carried out on a 1.1 μs production segment for each simulation, following a 400 ns equilibration period as in our previous study [25].