Molecular Insights Into Binding and Activation of the Human KCNQ2 Channel by Retigabine

FIGURE 1. Main conformational states, overall tetrameric structure, and RTG binding site of the KCNQ2 channel. (A) Schematic view of the RC and AO conformational states of the channel. The Pore and VSD domains are colored yellow and blue, respectively. The S4 and S4-S5 linker helices are shown as blue and green cylinders, respectively. (B) Top view of the tetrameric channel in the AC conformational state (PBD ID: 7CR0). (C) Zoom on the RTG binding site on the Pore domain of the KCNQ2 channel. The site is a crevice between two adjacent channel monomers (shown as orange and greenish yellow ribbons). Residues in contact with the drug in the experimental structure 7CR2 (Li et al., 2021) are shown using a CPK model with bright surfaces, while residues identified in this work based on previous knowledge are shown as sticks. Residues on the second monomer are labelled by primed numbers.

FIGURE 2. Conformations of the VSD domain. Top row: Key interactions established between charged/polar residues of the S1-S4 helices in the apo and RTG-bound experimental structures, followed by the three models described in this work. Helices S2, S3, and S4 are shown in dark yellow ribbons, while the sidechains of key residues are represented by sticks colored by atom type. H-bonds/salt bridges are indicated by black dotted lines. Light blue check marks identify interactions present in the apo experimental structure (7CR0) and reproduced in the homology models. Bottom row: Comparison among the molecular surfaces (bottom view from the intracellular side; monomers colored differently) of the channel in the experimental and modelled structures generate in this work.

FIGURE 3. Sampling of bound-like conformations of the RTG binding site (see Figure 1) during the MD simulations of unbound KCNQ2 in the three states considered in this work. The graph on the left displays the RMSD distributions (calculated on all heavy atoms of the residues lining the experimental RTG binding site) extracted from the corresponding MD simulations. The three pictures on the right of this graph show the structures featuring the lowest RMSD from the experimental geometry of the RTG site in each simulation. The S5, S6, and S5-6 linker helices from one monomer are shown as greenish yellow ribbons, while the S6 helix from the adjacent monomer (S6′) is colored in orange. Residues from each monomer are shown as sticks colored by atom type (carbon atoms colored as the ribbons). The experimental conformation is shown in grey color.

FIGURE 4. Effect of RTG binding on the flexibility of KCNQ2, measured in terms of average RMSF calculated on the stable MD simulations discussed in the main text after alignment of the Pore domain. The pictures on the left illustrate the increase in the RMSF values of the VSD domains after binding of RTG to the AO (upper panel) and RC (lower panel) states. The graphs on the right site help to better quantify such an increase.

FIGURE 5. Morphologies of the tunnels leading from the center to the cytoplasmic end of KCNQ2. (A,C,E) Tunnels (red surfaces) found in the main (A, C) and fourth (E) conformational clusters extracted from the cumulative trajectories of the channel in the AO state, AO bound to RTG, and RC bound to RTG, respectively. Each monomer is shown in ribbons colored differently, with the Pore domain solid and the rest of the protein transparent. The starting point set for tunnel detection is approximately indicated by a blue capital S letter. (B,D,F) Radii (r) vs. path length (l). The profile of the tunnel found in the most populated non-closed cluster (population in parenthesis) is shown by a black solid line, while the profiles associated to the tunnels found in the remaining clusters are shown by gray dashed lines. The total percentage of clusters bearing a tunnel is also reported in each graph.