Fernández-Mariño AI et al. (2018), Gating interaction maps reveal a noncanonical e...

XB-ART-56151

Nat Struct Mol Biol 2018 Apr 01;254:320-326. doi: 10.1038/s41594-018-0047-3.

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Gating interaction maps reveal a noncanonical electromechanical coupling mode in the Shaker K+ channel.

Fernández-Mariño AI , Harpole TJ , Oelstrom K , Delemotte L , Chanda B .

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Membrane potential regulates the activity of voltage-dependent ion channels via specialized voltage-sensing modules, but the mechanisms involved in coupling voltage-sensor movement to pore opening remain unclear owing to a lack of resting state structures and robust methods to identify allosteric pathways. Here, using a newly developed interaction-energy analysis, we probe the interfaces of the voltage-sensing and pore modules in the Drosophila Shaker K+ channel. Our measurements reveal unexpectedly strong equilibrium gating interactions between contacts at the S4 and S5 helices in addition to those between S6 and the S4-S5 linker. Network analysis of MD trajectories shows that the voltage-sensor and pore motions are linked by two distinct pathways: a canonical pathway through the S4-S5 linker and a hitherto unknown pathway akin to rack-and-pinion coupling involving the S4 and S5 helices. Our findings highlight the central role of the S5 helix in electromechanical transduction in the voltage-gated ion channel (VGIC) superfamily.

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	Figure 2. Interaction energy analysis residues in the intracellular gating interface(A) (Left panel) Side view of the F484 (orange) and R387 (purple) mapped on Kv 1.2/2.1 chimera structure. Only the S4–S5 and S6 domains of the same subunit are shown for clarity. (Right panel) Normalized Q-V curves of WT (grey squares), R387A (purple triangles), F484A (orange circles) and R387A-F484A (blue inverted triangles). (B) (Left panel) Side view of the S376 (red) and L382 (purple). The S376 residue is at the end of the S4 segment and the L382 is at the beginning of the S5 segment in the neighboring subunit. (Right panel) Normalized Q-V curves of WT (grey squares), L382A (purple triangles), S376 (red circles), S376A-L382A (blue inverted triangles). (C) (Left panel) Side view of the S376 (red) and Q383 (orange), which is at the start of the S4–S5 linker. (Right panel) Normalized Q-V curves WT (grey squares), S376 (red circles), Q383A (orange triangles), and S376A–Q383A (blue inverted triangles). Mesh (A–C) represents the surface based on the Van der Waals radii for selected atoms. Error bars represent SEM in all Q-V plots.
	Figure 3. Interaction energy analysis of residues in the transmembrane gating interface(A–D) (Top panels) Top-down view of the S4 and S5 helices from neighboring subunits obtained from the Kv1.2/2.1 chimera structure. (A and B) (Top panels) Top-down view of the S4 and S5 helices from neighboring subunits. Mapped in the structures are I372 (orange), L409 (red) and I405 (purple). (A) (Bottom panel) Normalized Q-V curves of WT (grey squares), I372A (orange circles), L409A (red triangles) and I372A-L409A (blue inverted triangles) (B) (Bottom panel) Normalized Q-V curves of WT (grey squares), I372A (orange circles) I405 (purple triangles) and I372A–I405A (blue inverted triangles). (C and D) Top-down view of the S4 and S5 helices from neighboring subunits. Mapped in the structure V369 (orange) and V408 (red) and S412 (purple). (C) (Bottom panel). Normalized Q-V curves of WT (grey squares), V369 (orange circles), V408A (red triangles) and V369A–V408A (blue inverted triangles). (D) (Bottom panel) Normalized Q-V curves which in addition to WT (grey squares) and V369 (orange circles) also shows S412 (purple triangle) V369A–S412A (blue inverted triangles). The mesh (A–D) represents the surface based on the Van der Waals radii for the residue within the helix, please note that only the mesh for the selected residue is shown.
	Figure 4. Long distance interactions between the S4 and the S4–S5 linker of the same subunit(A and B) (Top panels) Side view of S4 and S4–S5 linker from the same subunit of the Kv1.2/2.1 chimera structure. Mapped in the structure V369 (orange), S376 (red) and R387 (purple). (A) (Lower panels) Normalized Q-V curves of WT (grey squares), V369A (orange circles), S376A (red triangles) and V369A–S376A (blue inverted triangles). (B) Normalized Q-V curves of WT (grey squares) and V369A (orange circles), R387A (purple circles) and V369A–R387A (blue inverted triangles). The mesh (A and B) represents the surface based on the Van der Waals radii for the selected residue.
	Figure 5. Residue betweenness for pathways between S4 and S6 in the activated/open stateBetweenness is a measure of centrality of a residue in various allosteric pathways that link source and sink residues in that it calculates the number of shortest paths a residue is on. Residues with higher betweenness are hubs in the network and are therefore important for information flow along the network. Betweenness is calculated for residues of each individual subunit since in MD simulations, each subunit will evolve differently over time due to the stochasticity inherent in this process. (A–D) Each panel represents one of the four subunits where the subunit colors, source and sink residues were selected following the convention described in Fig. 1. Residue betweenness for each subunit is mapped on to the activated state structures. It is calculated using source residues where the c-α is, on average, within 9 Å of Arg 365 (R2). Residues with high betweenness are shown in dark red whereas low betweenness residues are shown in light red. In the top two panels showing subunits A and B, residues of high betweenness are within a single subunit and travel down S4 and along the S4–S5 linker finally linking up with the gate residues in the S6 subunit. In the bottom two panels showing subunits C and D, residues of high betweenness are on multiple subunits and travel from S4 to the neighboring subunit S5 then down the S5 helix. In these panels, the S6 helix of the neighboring subunit is also shown to identify the position of the sink residue. The intersubunit pathway remains consistent whether the sink residue is on the same or the adjacent subunit (Supplementary Fig. 4 G,H).
	Figure 6. Schematic showing the two modes of electromechanical coupling in a prototypical potassium channel(A) In the canonical mode, S4 (blue) acts a lever arm moving the S4–S5 linker (green) directly and thereby causing the lower half of the S6 helix (cyan) to readjust. In the resting state, the S4 helix is down which through the S4–S5 linker keeps the lower half of S6 in the closed state (left panel). When the S4 helix is up, the S4–S5 linker rotates upwards and allows the lower S6 helices to splay open (right panel). (B) Gear like movement of S4 helices directly shifts the position of the neighboring S5 helix. In this rack and pinion type of coupling, when S4 is in the resting state (left), it holds the S5 helix in a down position which forces the S6 gates to remain closed. The upward movement of the S4 helix (right panel), drives the S5 helix up and causes the S6 helices to open.

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase