Apo and ligand-bound high resolution Cryo-EM structures of the human Kv3.1 channel reveal a novel binding site for positive modulators

Fig. 1.. Structure of apo flWT-Kv3.1a. (A) Cryo-EM reconstruction of apo flWT-Kv3.1a. (B) Atomic model of apo flWT-Kv3.1a. The color code is identical in both figures and highlights the different domains and their organization in flWT-Kv3.1a. For clarity, only one monomer has been colored, the remaining three monomers are shown in light gray. The N-terminus end is colored in dark blue, the T1 domain in light blue, the T1-S1 linker in cyan, the VSD in light green, the S4–S5 linker in yellow, the PD in orange, and the C-terminus end in red. Potassium ions visible in the reconstruction were modeled and are colored in purple.

Fig. 2.. Comparison between the structures of Kv1.2–2.1 and flWT-Kv3.1a. (A) Overlay of a monomer of Kv1.2–2.1 paddle chimera structure (red, pdb code: 6ebk) with a tetramer of flWT-Kv3.1 (cyan, only one monomer colored). The overlay is done on the PD. (B) flWT-Kv3.1a and Kv1.2–2.1 structures are shown from an intracellular viewpoint to illustrate the 35° twisting motion. (C) Coordination site for Zn2+ atom (purple, and distances to nearest heavy atom reported in Angstroms) in flWT-Kv3.1a. The Zn-site appears to assist the T1 tetramerization by bridging His77 (T1), Cys104 (T1-S1 linker), and Cys105 (T1-S1 linker) from one monomer, with Cys83 (T1) from a neighboring monomer. (D) Zoom on the VSD. The electron density map and the corresponding atomic model are presented. R2, R3, R4, K5, and R6 residues within the S4 segment are presented in red. In the case of Kv3.1, R4 interacts with Phe256 within the charge transfer center in contrast to the structure of Kv1.2–Kv2.1 showing that R4 is located above Phe233, which interacts with K5. Phe233 in Kv1.2–Kv2.1 is the equivalent of Phe256 in Kv3.1.

Fig. 3.. Binding site observed for the Lu AG00563 positive modulator. (A) Cryo-EM density map (blue) with region of nonprotein density attributed to the ligand (yellow). (B) Close-up view of the ligand binding pocket, displaying both the ligand and protein experimental densities. (C) Simplified diagram of the protein–ligand interactions, highlighting the H-bond with Thr325, hydrophobic residues (green), and polar residues (cyan). (D) Tri-dimensional representation of interactions in the binding site. The S1 and S4 helices are shown in light green, S5 in orange, and the S4–S5 linker in yellow.

Fig. 4.. Comparison between the binding site of Lu AG00563 in flWT-Kv3.1a and the binding sites of PIP2 and RTG in Kv7.4. (A) Binding site discovered for Lu AG00563 at the interface between S1, S4, and the S4–S5 linker. The S1 and S4 helices are shown in light green, S5 in orange, and the S4–S5 linker in yellow. Lu AG00563 is shown in yellow. (B) Comparison with the Kv7.4 structure (pdb code: 7bym), highlighting similarities with the PIP2 binding site showing interactions with S1, S4, and the S4–S5 linker. In contrast, the RTG binding site is located next to the pore (S5) domain. PIP2 is shown in purple and RTG is shown in Cyan.