Molecular dynamics simulations of the calmodulin-induced α-helix in the SK2 calcium-gated potassium ion channel

Figure 1. Structural features of SK channels.A, schematic topological representation of an SK channel subunit (in gray except for helix hA in blue) in complex with CaM (red). B, cryo-EM structure of the Ca2+-free SK4-CaM complex, in which the CaM N-lobe is not visible. The CaM C-lobe is in red, and the helix hA-docking region is in blue (PDB: 6CNM (11)). C, crystallographic structure of the SK2 CaM-binding domain (CaMBD) (PDB: 1G4Y (14)) with the C-lobe in red and the helix hA-docking region in blue. D, solution structure of an SK2 peptide with the helix hA sequence, with the binding region colored in blue (PDB: 1KKD (18)). CaM, calmodulin.

Figure 2. Initial SK2 conformations and secondary structure analysis, according to the DSSP definition (21), calculated along a Hamiltonian replica exchange molecular dynamics trajectory.A, amino acid sequence of the studied SK2 fragment and cartoon depictions of the 1KKD conformation (18) (with the T430-I433 segment as an α-helix, one turn) and one of the final snapshots in our previous work (20) (with the E429-N436 segment as an α-helix, two turns). B, secondary structure content in aqueous solution and in the absence of CaM, starting from the 1KKD (“one turn”) conformation. C, secondary structure content in complex with the CaM C-lobe, starting from the 1KKD (“one turn”) conformation. D, secondary structure content in complex with the CaM C-lobe, starting from the “two turns” conformation. CaM, calmodulin.

Figure 3. Initial and final simulation snapshots, starting from the 1KKD structure (“one turn”, left) and from one of the final snapshots in our previous work (20) (“two turns”, right). The SK2 calmodulin-binding domain rigid fragments (N421-T437) are displayed in blue; the CaM C-lobe in red; and the Ca2+ ions, as yellow van der Waals spheres. CaM, calmodulin.

Figure 4. Mobility, solvent exposure, and SK2-CaM contacts calculated along a Hamiltonian replica exchange molecular dynamics trajectory.A and B, root-mean-square fluctuation (RMSF) and solvent-accessible surface area (SASA), respectively, of each residue in the rigid fragment of the SK2 CaM-binding domain (N421-T437), calculated along a Hamiltonian replica exchange molecular dynamics trajectory starting from either the T430-I433 (“one turn”) or the E429-N436 (“two turns”) segment as an α-helix. C and D, contacts between L427 or W431 and the CaM C-lobe, calculated along these two trajectories. The indicated CaM residues are those whose average distances to either L427 or W431 are at most 0.5 nm. CaM, calmodulin.

Figure 5. Orientation of the W431 side chain. Time evolution of the angle determined by the C-lobe of CaM, the Cα of W431, and its aromatic ring (as shown on the right side) for both the “one turn” (A) and the “two turns” (B) systems. CaM, calmodulin.

Figure 6. Secondary structure analysis of the simulations starting from two alternative conformations, with W431 rotated by 180 degrees.A, starting structure of the “one turn” simulation with W431 pointing outward (i.e., toward the solvent, top) and of the “two turns” simulation with W431 pointing inward (i.e., toward the C-lobe of CaM, bottom). B–D, same as in Figure 2, but the simulations in the presence of the C-lobe of CaM were started from the conformations shown in A. CaM, calmodulin.

Figure 7. Mobility, solvent exposure, and SK2-CaM contacts calculated along the alternative Hamiltonian replica exchange molecular dynamics trajectory. Same as in Figure 4, but the simulations were started with W431 rotated by 180 degrees, so that the side chain of W431 was initially pointing toward the opposite direction: outward (i.e., toward the solvent) in the “one turn” system and inward (i.e., toward the C-lobe of CaM) in the “two turns” system. A and B, root-mean-square fluctuation (RMSF) and solvent-accessible surface area (SASA), respectively, of each residue in the rigid fragment of the SK2 CaM-binding domain (N421-T437), calculated along the alternative Hamiltonian replica exchange molecular dynamics trajectory starting from either the T430-I433 (“one turn”) or the E429-N436 (“two turns”) segment as an α-helix. C and D, contacts between L427 or W431 and the CaM C-lobe, calculated along these two trajectories. The indicated CaM residues are those whose average distances to either L427 or W431 are at most 0.5 nm. The inset in D shows the distances between V426 or W431 and CaM C-lobe residues averaged only over the fully helical structures. CaM, calmodulin.

Figure 8. Schematic depiction of the picture emerging from our simulation results. To induce an α-helix along the whole CaMBD of SK2, CaM would select those SK2 conformations with at least two α-helical turns, where W431 is less mobile, and then induce additional α-helical structure through interactions with V426 and L427. CaM, calmodulin; CaMBD, calmodulin-binding domain.

Figure 9. Schematic depiction of the workflow followed to build the initial conformations of the simulated complexes between the SK2 CaMBD and the Ca2+-loaded C-lobe of CaM. CaM, calmodulin; CaMBD, calmodulin-binding domain.