Fig. 6: Binding site localization and in silico simulation results.

a Depiction of tetrameric KCNQ1 AO model with subunits colored in magenta (Mol A), yellow (Mol B), green (Mol C) and blue (Mol D). Proposed binding site of (R)-L3 is marked by red square. b Close-up depiction of (R)-L3 (CPK-color coded with cyan for carbon) binding site with S4 (yellow) and S4S5 linker (orange) from subunit Mol B and S5 and S6 helix (magenta) from subunit Mol A. Previous analyzed amino acids from S5 and S6 with impact on (R)-L3 activity are colored in purple, while newly analyzed residues from S4 and S4S5 linker are colored in green or violet. c Schematic illustration of (R)-L3 binding site between S4 (yellow) and S4S5 linker (orange) from subunit Mol B and S5 and S6 (pink) from subunit Mol A. d Depiction of KCNQ1 AO model embedded in membrane for MD simulation. Subunits Mol A-D are colored like in 5A and (R)-L3 binding site is marked by red square. e Root mean square deviation (RMSD) of 30 ns MD simulations for complete modeled structures AO (n = 5 simulations), AC (n = 3) and RC (n = 3) with (R)-L3 for every simulation. Means are shown as bar and have no significant differences indicated by ns. f RMSD of Ligand movement of (R)-L3 from start to end of the 30 ns MD simulation of KCNQ states AO (n = 5), AC (n = 3) and RC (n = 3). g (R)-L3 Root mean square fluctuation (RMSF) for 30 ns MD simulation with KCNQ1 state models AO (n = 5), AC (n = 3) and RC (n = 3). Mean differences are not significant (ns; p > 0.05). h Binding Energy [kJ/mol] of (R)-L3 calculated for all simulation snapshots from 10 to 30 ns of MD simulations for KCNQ1 models AO (n = 405 snapshots), AC (n = 243) and RC (n = 243). i Percentage duration of hydrophobic interaction between (R)-L3 and mutated amino acids from S4 and S4S5 linker over total MD simulation time of 30 ns for AO simulations (n = 5). j, k Backbone RMSD and RMSF of mutated residues in the absence (n = 3 simulations)/presence (n = 5 simulations) of (R)-L3. l activity of 1 µM (R)-L3 at wildtype KCNQ1 expressing oocytes compared to mutations W248F, W248A, and W248R (n = 4 for WT, n = 5 for W248F, W248A, and W248R).