Mutation‐Induced Pocket Deactivation: How Ser353/Pro245 Alters K Ca 2.2 vs. K Ca 3.1 Ligand Selectivity

Figures

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  Figure 1. (A) Structure of the Compound 4‐bound KCa2.2 channel (green ribbons, PDB ID: 9O5O [6]), showing Compound 4 located in the S5‐Phelix‐S6 pocket. Co‐determined K+ ions located in the ion selectivity filter, Compound 4, gating residue Val390 and co‐determined Ca2+ ions are represented with Van der Waals spheres. For better clarity, the represented Compound 4, residue Val390 and Ca2+ ions correspond to one subunit only. (B) Chemical structure of the KCa2.2 inhibitor Compound 1 and the KCa2.2 activator Compound 4 [6].

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  Figure 2. S5‐Phelix‐S6 pocket in KCa2.2 (green ribbons and sticks, PDB ID: 9O53 [6]) and KCa3.1 (orange sticks, PDB ID: 6CNM [5]). Compound 1 is represented with pink sticks, while Compound 4 with light blue sticks. Residues are indicated using the one‐letter code, with first residues from KCa2.2 followed by the ones from KCa3.1. (A) Amino acid substitutions between the KCa2.2 and KCa3.1 channels. (B) Clash between residue Trp216 in KCa3.1 and Compound 1/Compound 4; (C) Focus on the clash between Trp322 (green Van der Waals spheres) and the mutated Ser353Pro residue (purple Van der Waals spheres) in structure KCa2.2. (D) Alignment of KCa2.2 and KCa3.1 channel sequences. Residues lining the S5‐Phelix‐S6 pocket (within 5 Å from Compound 1) are highlighted in orange. Residue substitutions between KCa2.2 and KCa3.1 are indicated with a red box. The sequence alignment was computed using the EMBL‐EBI Clustal Omega web server [30].

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  Figure 3. Plots of the χ 1 and χ 2 dihedral angles of residue Trp216 during the KCa3.1 MD simulations. Each dot represents a combination of dihedral angles in a specific frame of the trajectory. Time evolution along the simulation is represented by colour shift from yellow to green. Reference dihedral angles obtained from the Compound 4‐bound KCa2.2 and KCa3.1_open cryo‐EM structures are indicated with a blue and red cross, respectively. (A) System KCa3.1_closed_WT, run 1, chain A. (B) System KCa3.1_closed_P245S, run 1, chain A. (C) System KCa3.1_open_WT, run 1, chain A. (D) System KCa3.1_open_P245S, run 2, chain B.

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  Figure 4. Interactions established by residue Trp216 during the KCa3.1_open MD simulations. 3D representation of the interactions established by the Trp216 side chain in (A) conformation a, (B) conformation b, and (C) conformation c. The KCa3.1 channel is represented by green ribbons, with residues in green sticks. The different Trp216 conformations are highlighted in purple, orange and cyan, respectively. Bar plots representing the time evolution of the interactions established by Trp216 across (D) the KCa3.1_open_WT simulations (run1, chain A), and (E) the KCa3.1_open_P245S simulations (run1, chain B). Non‐covalent interactions are indicated with dashed lines, specifically H‐bonds are represented in cyan, hydrophobic interactions in yellow and π‐π stacking in magenta.

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  Figure 5. (A) Volume variation of S5‐Phelix‐S6 binding pocket across the KCa3.1_open_P245S simulation (run2, chain B). (B) Comparison between the Compound 4 pose obtained via ensemble docking (gold sticks) and the reference cryo‐EM binding mode (grey sticks). The KCa3.1 channel is represented by green ribbons, with residues in green sticks. Non‐covalent interactions are indicated with dashed lines, specifically H‐bonds are represented in cyan, hydrophobic in yellow and π‐π stacking in magenta.