2.6 | Molecular dynamics simulations
The selected poses in the side pocket were triplicated in the other side pockets. The selected pose in the central cavity was also included forming a complex with five ligands, four in each side pocket and one in the central cavity. The protein-ligands complexes were embedded into a pre-equilibrated POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) bilayer, then the systems were solvated using the SPC (single point charge) water model. K+ ions were placed at sites S2 and S4 of the selectivity filter and water molecules at sites S1 and S3. Cl- ions were added to neutralize the system. An ion concentration of 0.15 M KCl on both sides of the channel was set for the MDs. The systems were equilibrated by 20 ns in NPT ensemble with positional restraints of 1.0 kcal*mol-1-2 on the secondary structure of the protein, ligands, and ions at the selectivity filter. Temperature and pressure were kept constant at 300 K and 1.01325 bar, respectively by coupling to a Nose-Hoover Chain thermostat (Cheng & Merz, 1996) and Martyna-Tobias-Klein barostat (Martyna, Tobias et al. , 1994) with an integration time step of 2 fs. Later, positional restraints were removed, and a molecular dynamics simulation of 100 ns was done per system using an NPγT (semi-isotropic ensemble) with constant surface tension of 0.0 bar Å as production. Molecular dynamics simulations were performed used Desmond (Bowers, Chow et al. , 2006) software, v2019-1 and OPLS2005 force field (Shivakumar, Williamset al. , 2010). The same MDs protocol was applied to the Kv1.5 protein (without ligands) as control. From the 100 ns-MDs of production, 200 structures from the last 20 ns (every 0.1 ns) were selected to perform the clustering analysis of both ligands (S-bupivacaine and ropivacaine) in each binding site. Protein structure of the selected frames were aligned, and then the cluster analysis was performed by selecting the heavy atoms of the ligand with a defined RMSD cut off = 2 Å. Clusters were calculated using the Clustering v2.0.1 plugin (Github June 2014) in the VMD software (University of Illinois, Illinois, USA).