MD simulations of the Hsp90 structures (each of 20 ns duration) were performed for a closed ATP-bound conformation of yeast Hsp90 (crystal structure) (PDB ID 2CG9) [54] (link); a “V-shaped” conformation of the mammalian Grp94 homologue from complexes with ADP (PDB ID 2O1V) and AMP-PNP (PDB ID 2O1U) (crystal structure) [56] (link); an open Apo form of the bacterial homologue HtpG (PDB ID 2IOQ) (crystal structure) [55] (link); a semi-closed, ADP-bound form of the bacterial homologue HtpG (PDB ID 2IOP) (crystal structure) [55] (link); and an extended Apo HtpG conformation (solution structure from SAXS studies) [61] (link). All crystallographic water molecules, bound inhibitors, and other heteroatoms were removed. The retrieved structures were examined for missing and disordered residues. The missing residues and unresolved structural segments were modeled using the program MODELLER which is an automated approach to comparative protein structure modeling by satisfaction of spatial restraints [153] (link), [154] (link). MD simulations were carried out using NAMD 2.6 [155] (link) with the CHARMM27 force field [156] (link), [157] and the explicit TIP3P water model as implemented in NAMD 2.6 [158] . The VMD program was used for the preparation and analysis of simulations [159] (link), [160] (link). The employed MD protocol was described in full details in our earlier studies [161] (link)–[163] (link). In brief, structures were solvated in a water box with the buffering distance of 10 Å. Assuming normal charge states of ionizable groups corresponding to pH 7, sodium (Na+) and chloride (Cl−) counter-ions were added to achieve charge neutrality in MD simulations at physiological concentration of 0.15 mol/L. All Na+ and Cl− ions were placed at least 8 Å away from any protein atoms and from each other. The system was subjected to initial minimization for 20,000 steps (40 ps) keeping protein backbone fixed which was followed by 20,000 steps (40 ps) of minimization without any constraints. Equilibration was done in steps by gradually increasing the system temperature in steps of 20 K starting from 10 K until 310 K and at each step 15000 steps (30 ps) equilibration was run keeping a restraint of 10 Kcal mol−1 Å−2 on protein alpha carbons (Cα). Thereafter the system was equilibrated for 150,000 steps (300 ps) at 310 K (NVT) and then for further 150,000 steps (300 ps) at 310 K using Langevin piston (NPT) to achieve uniform pressure. Finally the restrains were removed and the system was equilibrated for 500,000 steps (1 ns) to prepare the system for simulation. An NPT simulation was run on the equilibrated structure for 20 ns keeping the temp at 310 K and pressure at 1 bar using Langevin piston coupling algorithm. The integration time step of the simulations was set to 2.0 fs, the SHAKE algorithm was used to constrain the lengths of all chemical bonds involving hydrogen atoms at their equilibrium values and the water geometry was restrained rigid by using the SETTLE algorithm. Nonbonded van der Waals interactions were treated by using a switching function at 10 Å and reaching zero at a distance of 12 Å. The particle-mesh Ewald algorithm (PME) as implied in NAMD was used to handle long range electrostatic forces.
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