Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel

Figures

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  Figure 1.. Simulation of a model of the pore domain of the Shaker channel with partially open intracellular gate.(A) A Shaker model was built up based on the pore domain of the crystal structures (PDB accession no. 2R9R) of Kv1.2/Kv2.1 by mutating a few nonconserved residues (cyan). The nonconserved residues are also highlighted in cyan box in sequence alignment. Based on previous results (Li et al., 2018), the width of 18 Å measured by the Cα–Cα cross-subunit distances of S479 (the equivalent residue of T112 in KcsA) in the 2R9R x-ray structure suggests that it most likely corresponds to a partially open structure. (B) Time series of the cross-subunit distance (r) between the Cα atoms of G444 of diagonally opposed monomers A and C (green) and B and D (red) for six simulations started from the partially open Shaker model. Trajectories (Traj.) 1–3 were started from conductive filter without restraint. In all three trajectories, the filter remained in the conductive conformation. Trajectories 4–6 were initially restrained with their filter in the constricted conformation (highlighted in gray), in which the filters all returned to the conductive conformation once the restraints were released.…

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  Figure 2.. TMD simulation drives the intracellular gate of the Shaker channel open progressively in a stepwise manner.(A) Overlap the Kv1.2/Kv2.1 structure with KcsA structures, to show how to trigger further opening of the intracellular gate by using two KcsA structures (PDB accession nos. 3F7V and 3F5W) as references. (B) Stepwise bias MD simulations to trigger gate opening. The first 100-ns simulation is performed by using 3F7V as a target reference. The second 100-ns simulation is performed by using 3F5W as target reference. dist, distance.

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  Figure S1.. Differential conformational preferences of the Shaker channel with different opening degrees. (A) 15 snapshots (red dot) were selected from the bias MD trajectory as initial seeds for unbiased MD simulations. The initial seeds were clustered into six groups (circled) based on the opening degree. (B) The probability density histograms of the G444 Cα–Cα distance of two pairs of diagonally opposed monomers A and C (red) and B and D (blue) in 15 unbiased MD simulations with opening degree of the intracellular gate restrained. dist, distance; prob, probability.

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  Figure 3.. MD simulation of the Shaker channel with an opening of 32 Å for the intracellular gate yields a stable constricted conformation of the selectivity filter. The four channel subunits are labeled A, B, C, and D in clockwise order seen from the extracellular side. (A) Representative conformation of the selectivity filter and water molecules bound behind the filter. (B) Time series of the number of water molecules occupying the water-binding bottom site within each subunit (upper and middle) and the cross-subunit distance between the Cα atoms of G444 of diagonally opposed subunits A and C (lower left) and B and D (lower right) during the 22-µs simulations in the wide-open channel.

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  Figure S2.. Simulations of the constricted conformation generated using the AMBER force field with the PMEMD MD engine of AMBER. Evolution of the constriction starting from the CHARMM-generated conformation. The AMBER-compatible topology and coordinates file were produced by the CHARMM-GUI's force field converter, and the simulation was performed using the AMBER scripts provided by CHARMM-GUI. dist, distance; FF, force field.

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  Figure 4.. 2-D–PMF to assess the conformational preferences of the selectivity filter with partially and wide-open intracellular gates based on a model of the pore domain of the Shaker channel. The horizontal reaction coordinate r describes the width of the selectivity filter and is defined as the average cross-subunit pinching distance between the Cα atoms of G444. The vertical reaction coordinate z indicates the position of the external K+ ion along z axis relative to the center of the selectivity filter. The lower panel is the 1-D–PMF along horizontal reaction coordinate r, with integration of the vertical reaction coordinate z. The typical conformations for two free energy basins (A and B) are shown in stick representation for protein and van der Waals representation for both water and K+ ions. The conductive filter conformation is the most stable when the gate is partially open (18 Å) as in the x-ray structure PDB accession no. 2R9R. The constricted filter conformation is the most stable when the gate is wide open (32 Å) as in the x-ray structure PDB accession no. 3F5W. The 2-D–PMF with a gate opening of 18 Å previously appeared in (Li et al., 2021).

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  Figure 5.. Different constricted structures for Shaker and KcsA. Top: Sequence alignment of Shaker and KcsA for the pore helix and the selectivity filter; 15 residues are identical out of a total of 23 residues. Below the sequence alignments are shown typical constricted conformations of the filter, with a top view (left column) and side view (middle column), as well as 2-D water occupancy map (right column) for different channels. Those are WT KcsA (first middle panel), KcsA E71V mutant (second middle panel), and Shaker (bottom). The top view of the filter only shows water molecules in bottom site of four subunits, and the backbone of V76 and G77 in KcsA (or the equivalent V443 and G444 in Shaker). For the 2-D occupancy map of the water molecules behind the selectivity filter, the horizontal reaction coordinate ρ is defined as the distance of the oxygen atom of water molecule with the center of the selectivity filter along the x-y plane. The vertical reaction coordinate z indicates the position of water molecules along the z axis relative to the center of the selectivity filter.

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  Figure S3.. 2-D–PMF explicitly along the two distances between the Cα atoms of G444 of diagonally opposed subunits A and C (r1) and B and D (r2) for the Shaker channel at 32 Å opening.

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  Figure S4.. A stable constricted conformation of the selectivity filter in the KcsA E71V mutant. Top: Representative conformation of the selectivity filter and water molecules bound behind the filter. Bottom: Time series of the number of water molecules occupying the water-binding bottom site within each subunit (upper and middle panel) and the cross-subunit distance between the Cα atoms of G77 of diagonally opposed subunits A and C (lower left) and B and D (lower right) during the 21-µs simulations in the wide-open channel.

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  Figure 6.. 2-D–PMF to assess the conformational preferences of different constricted structures. The horizontal reaction coordinate r describes the width of the selectivity filter and is defined as the average cross-subunit pinching distance between the Cα–Cα atoms of G444 in Shaker or G77 in KcsA. The vertical reaction coordinate Ψ indicates the backbone Ψ dihedral angle of the valine in the center of the selectivity filter. The typical conformations for free energy basins are shown in stick for protein and van der Waals representation for water molecules from the top view.

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  Figure 7.. A stable conformation constricted at the extracellular S0 site of the selectivity filter in a model of the pore domain of the W434F Shaker mutant.(A) Time series of the cross-subunit distance between the Cα atoms of diagonally opposed subunits, respectively, for G446 to monitor the conformation of S0 site (upper) and G444 to monitor the conformation of S2 site (middle), and the RMSD of the selectivity filter backbone (lower) by using the initial (blue) or last (red) conformation as reference. (B) Side views of the last snapshot of the selectivity filter at the end of the 10-µs trajectory for the subunits A and C (left) or B and D (middle) of the W434F Shaker mutant. A scheme (right) is shown to highlight the selectivity filter conformation constricted at the S0 (G446) site. (C) Side views of the selectivity filter for constricted Shaker WT (left) and WT KcsA (middle). Both are constricted at the S2 site as shown in the scheme (right). (D) Side views of the selectivity filter for conductive Shaker WT (left), WT KcsA (middle), and the scheme (right). The simulation of the W434F mutant was performed with a model with an intracellular gate opening of ∼18Å.

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  Figure 8.. The rotameric redistributions of key residues responsible for allosteric coupling upon gate opening in both Shaker and KcsA. (A–D) The rotamer density histograms of key residues for multiple ensembles to represent the different opening degrees from partially to wide open of the intracellular gate. The simulations for Shaker are trajectories 8–23 (Table S1), and the trajectories for KcsA are trajectories 1–10 in a previous study (Li et al., 2018). (E and F) Typical structures to show the process from partially to wide open of the intracellular gate for Shaker (left) and KcsA (right). prob, probability.