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

In collaboration with Lundbeck, leadXpro solved the first structure of a human Kv3 channel in complex with a positive modulator.

The cryo-EM structures of Kv3.1 at 2.6 Angstrom, in the apo state and in a complex with a positive modulator designed by Lundbeck (Lu AG00563) represent the first structure of the Kv3 channel family of drug targets, of which many members are implicated in diseases, in particular in the CNS space.

Figure 1: Left: Cryo-EM reconstruction of apo flWT-Kv3.1. Right: Atomic model of apo flWT-Kv3.1. The color code is identical in both figures and highlights the different domains and their organization in flWTKv3.1.
Figure 1: Left: Cryo-EM reconstruction of apo flWT-Kv3.1. Right: Atomic model of apo flWT-Kv3.1. The color code is identical in both figures and highlights the different domains and their organization in flWTKv3.1.

Of particular interest is the discovery of a zinc binding site that has been hypothesized to participate in the channel regulation. The zinc atom is seen to stabilize a rotated conformation by 35 deg of the cytoplasmic T1 domain which is not seen in other Kv classes.

Figure 2: T1 cytoplasmic domain adopting a 35° twisted conformation. A. Overlay of a monomer of Kv1.2-2.1 paddle chimera structure (red, pdb code: 6ebk) with a tetramer of apo flWT-Kv3.1 (cyan, only one monomer colored). The overlay is done on the pore domain. B. apo flWT-Kv3.1 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 flWTKv3.1. 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.
Figure 2: T1 cytoplasmic domain adopting a 35° twisted conformation. A. Overlay of a monomer of Kv1.2-2.1 paddle chimera structure (red, pdb code: 6ebk) with a tetramer of apo flWT-Kv3.1 (cyan, only one monomer colored). The overlay is done on the pore domain. B. apo flWT-Kv3.1 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 flWTKv3.1. 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.

In addition, the binding site identified for Lu AG00563 was found to be unique in all related Kv structures and located between the voltage sensory domain and the channel pore, in a region known to be a hotspot for disease-causing mutations. The knowledge of such a site for positive modulators could now set the stage for future structure-based drug discovery programs.

6643_Fig3AB
Figure 3: Binding site observed for the Lu AG00563 positive modulator. A. Cryo-EM density map (blue) with region of non protein 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.
Figure 3: Binding site observed for the Lu AG00563 positive modulator. A. Cryo-EM density map (blue) with region of non protein 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.

Reference:

Mathieu Botte, Sophie Huber, Denis Bucher, Julie K Klint, David Rodríguez, Lena Tagmose, Mohamed Chami, Robert Cheng, Michael Hennig, Wassim Abdul Rahman: Apo and ligand-bound high resolution Cryo-EM structures of the human Kv3.1 channel reveal a novel binding site for positive modulators, PNAS Nexus, Volume 1, Issue 3, July 2022, pgac083, https://doi.org/10.1093/pnasnexus/pgac083