Allosteric Mechanisms Underlying Long QT Syndrome Type 2 (LQT2)-Associated Mutations in hERG Channels

Figures

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  Figure 1:. Representations of the KV11.1 channel encoded by the KCNH2 gene. Two monomers out of four are shown for clarity. Transmembrane helices S1-S5 are labeled in one monomer of the tetrameric channel. The N- and C-terminal cytosolic PAS (pink) CLINKER (purple) and CNBD (lilac) regions are labeled. The variants considered in this study are rendered in the subpanel relative to the selectivity filter. Non-trafficking variants (Class II) are depicted in black spheres, while non-conducting variants are depicted in red spheres..

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  Figure 2:. Building Molecular Dynamics systems of KV11.1 channel a) Top-view and side-view of the tetrameric structure of KV11.1 channel domains (VSD in gray, S4 in blue, S4-S5LINKER in pink, S5 in orange, pore domain in cyan, CLINKER in purple and CNBD in lilac). Residues that will be mutated according to Class II and Class III missense mutations are depicted in black and red spheres, respectively. b) KV11.1 structure embedded into POPC bilayer and two slabs of water molecules. Ions are omitted for clarity. c) Two K+ ions were placed in the SF. Only two opposing subunits were shown for clarity..

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  Figure 3:. Average pore size of KV11.1 channel variants over triplicate MD simulations Panels (a), (c) and (e) show average radius profiles of the entire pore domain in all KV11.1 variants, the SF in R534 mutants, and the Cryo-EM structures of wild-type KV11.1 in high and low [K+] environments [37], respectively. In panel (c), red areas represent positions occupied by carbonyl oxygen atoms of each SF residue throughout the wild-type KV11.1 MD trajectory. Panels (b), (d) and (f) depict the average accessible solvent surface of the KV11.1 R534L variant according to the two different V625 carbonyl peptide bond orientations (relative to the conduction pathway center) and the wild-type KV11.1. K+ ions in the pore are ommitted for clarity. Pore surfaces are colored according to calculated average pore radii. Values below 1.33 Å (K+ ionic radius) are colored red. Values ranging from 1.33 Å to 3.6 Å (K+ hydrodynamic radius) are colored green. Values above 3.6 Å are colored blue..

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  Figure 4:. Ramachandran plots of selectivity filter residues in KV11.1 variants (a) Cartoon representation of KV11.1 pore domain in the wild-type (left) and enlarged views of KV11.1 selectivity filters in the wild-type, the single mutant R534L (framed in red) and the double mutant R534L-Y652C. In each panel, the pore domain is colored in cyan. The sidechain of PHELIX residue S620 (pink) and the main chain of SF residues T623 (lilac), S624 (black), V625 (yellow), G626 (lime green), F627 (green), and N629 (cyan) are represented in sticks. (b) Ramachandran plots of the Φ/Ψ torsion angle values (in degrees) of PHELIX S620 and SF residues T623, S624, V625, G626, F627 and N629 in the MD trajectories of KV11.1 wild-type (left), R534L (middle, framed in red), and R534L-Y652C (right) variants. The area occupied by the Φ/Ψ values for V625 and G626 in the MD trajectory of the non-trafficking KV11.1 mutant R534L is framed in red..

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  Figure 5:. Identifying KV11.1 trafficking-dependent interaction in the pore domain (a) Cartoon representation of the KV11.1 pore domain in the wild-type (WT). (b) Enlarged views of selectivity filters of KV11.1 wild-type (left), single mutant R534L (middle), and double mutant R534L-Y652C (right). (c) Probability density functions of the interatomic distance between the V625 backbone oxygen and S620 side chain oxygen for every subunit (chains A-D) of wild-type and variants R534C, R534L, and R534-Y652C. The 3.5 Å threshold characterizing hydrogen bonds is represented by a vertical dashed red line. (d) Table reporting the percentage of the area under the probability density curves falling below 3.5 Å obtained in panel (b) throughout the MD trajectories of all KV11.1 variants..

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  Figure 6:. Identifying KV11.1 trafficking-dependent interactions in the pore (a) Sequence alignment of transmembrane helices S2 and S4 in Voltage-Gated K+Channel homologs KV7.1, Shaker, KV1.2, KV2.1, and KV11.1 (black frame). The conserved charge transfer center (CTC) F463 is framed by a red dashed line. (b) Structural mapping of conserved S2 (solid gray), S3 (transparent gray), and S4 (blue) residues and interactions in the wild-type model (left) and WT-like double mutant D466K-R534D (right). Residues are shown as sticks; acidic residues are red, basic residues are blue. The CTC is colored gray. (c) and (d) show the structural mapping of S2-S4 interactions in KV11.1 R531 (R531Q, R531W) and R534 (R534C, R534L) variants, respectively. Color coding matches panel (b)..

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  Figure 7:. Correlation matrices of pore domain residue interhelical interactions in wild-type and non trafficking KV11.1 variants (a) Pearson coefficient heatmap of interatomic distances of pore domain residue pairs in MD trajectories of (a) wild-type KV11.1 and (b) KV11.1 non-trafficking mutants R534C and R534L. For each residue pair, the Pearson coefficient was calculated with p ¡ 0.05; blank areas represent non-significant values. The emergence of high-correlation clusters (red/blue) in the non-trafficking variants reveals an abnormal allosteric pathway where the selectivity filter becomes mechanically locked to the cytoplasmic CLINKER assembly. For both panels, two horizontal bars are colored according to the region of each residue in the corresponding residue pairs..