Figure 1.
RMSD analysis of ED opening simulations.
A) Average of the backbone RMSD (without loops) of ten opening ED simulations. The open crystal structure was used as reference. The standard deviation is indicated by error bars. B) Comparison of the average structure (built out of the minimal RMSD structures of the ten ED simulations; yellow to red) and the open crystal structure (gray). The RMSD of the Cα atoms is shown as a spectrum from yellow to red. For the sake of clarity, only the two opposite SUs are shown.
Figure 2.
Pore radius profiles derived from backbone atoms of channel states.
A) 3D representation of the pore domain depicting the HOLE profile. For the sake of clarity, only two opposing SUs are shown. B) Comparison of the profiles formed by the closed (red dashed line), intermediate (blue dashed line), and open (green dashed line) crystal structures with the average of the ten ED simulation structures (black dashed line). The subtle differences of the ED simulation structures in the activation gate region are indicated as standard deviation by error bars.
Figure 3.
Conformational changes of F103 during activation gate opening and SF conformations of channel states.
A) Analysis of χ1 angle dynamics of F103 (in ten opening ED simulations). Changes of the F103 orientation (χ1 angle) were measured over time. An angle of −70° indicates the “up” state (blue) while an angle of −180° represents the “down” state (yellow). The percentage of state was calculated from the end states at 20 ns of the ten ED simulations. B) SF and P-helix of the closed crystal structure (gray). Blue spheres represent K+ ions. C) SF and P-helix at the end of the 20 ns ED simulation structure with deprotonated E71 (yellow). D) SF and P-helix at the end of the 20 ns ED simulation structure with protonated E71 (yellow). E) SF and P-helix of the open inactivated crystal structure (gray). The G77 conformation defines the SF state as it was shown by Cuello et al. [12].
Figure 4.
Free energy profile of gate opening.
The blue, green, and yellow shades depict the sampling of the closed, intermediate, and open structures along the first EV. A) Free energy profile of the activation gate opening derived from 3.9 µs umbrella sampling simulations. The X marks indicate the positions of the crystal structures. B) χ1 angle dynamics of all four F114 during activation gating. C) Distance between opposite T112 as a measure of pore opening.
Figure 5.
Analysis of χ1 angle dynamics of F114 and influence on packing.
A) Percentage of F114 in the up (blue) and down (yellow) state over time. Packing of the F114 (transparent orange spheres) in the closed conformation (B), the transition state (C), the open states with F114 in the up (D) and the down state (E). Amino acids interacting in all states are shown in green. Interacting amino acids in the closed/open/transition state are represented in blue/yellow/red.
Figure 6.
Lipid interactions of TM2 helices during activation gate opening.
A) Average number of H-bonds between H-bond forming residues (W113 and R117) of the C-terminal TM2 helices and lipid head groups was measured over time. B) Average number of H-bonds of R117 with lipids. C) Average number of H-bonds of W113 with lipids. D) Representation of one SU in the closed (light blue) and open (marine blue) conformation with lipids. H-bond forming residues W113 and R117 are shown as yellow sticks. Lipids are depicted as gray lines while phosphate groups are shown as orange spheres. Dashed black lines represent H-bonds.
Figure 7.
RMSD of cooperativity simulations.
ED was applied on one (A), two (B), and three (C) SUs. The open structure was used as reference. For simulations with ED applied on one SU and two SUs, only one simulation each was performed. For ED simulations on three SUs, the average of the backbone RMSD without loops of ten simulations was measured. Standard deviation is indicated by the error bars.