Generation of hERG channel models in the closed (a), open (b), and inactivated (c) states.

The lower limit of the pore radius color profile (1.15 Å) indicates the minimum radius to accommodate a water molecule, and the upper limit (2.30 Å) indicates sufficient space to fit two water molecules side-by-side. “Max seq” is a setting in ColabFold that denotes the maximum number of cluster centers and extra sequences that the MSA used for AlphaFold2 will be subsampled to. “# models” indicates the number of models predicted using the provided structural templates.

Clustering of AlphaFold2-predicted hERG channel models.

a) Clusters created from 100 models predicted for each state. Each structure visualized is colored according to the per-residue confidence metric (pLDDT). The closed-state models are clustered based on the C-alpha RMSD of the entire protein models. The inactivated- and open-state models are clustered based on the all-atom RMSD of the selectivity filter (S624 – G628). To represent each cluster, the top 5 models ranked by average pLDDT are shown. The bar graphs display the mean pLDDT values for the clustered segment across all models within each cluster, accompanied by the standard deviation. Clusters containing less than three models are categorized as outliers. b) The models chosen for subsequent analysis.

Structural comparison of different hERG channel state models.

a) Visual comparison of the closed, open-, and inactivated-state models. b) Pore radius for the SF and drug binding region (upper) and for the entire pore (lower). c) Comparison of the VSD conformation in each model, showcasing the positively charged Arg and Lys gating-charge residues (yellow), located on the S4 helix, and the gating charge transfer center residue, F463 (magenta), on the S2 helix. d) Distances between the Cα atom of F463 to the Cα atom of each of the gating-charge residues.

Criteria for different type of non-bonded interactions used in analyses

Mutations known to affect hERG channel inactivation.

Movement of K+ ions through hERG SF.

The z coordinates of K+ ions are tracked as they traverse the pore of the channel from the intracellular gate (lower y-axis limit) to the extracellular space (upper y-axis limit) under the membrane voltage of 750 mV. Putative K+ binding sites in the SF (S0 – S5) are marked using blue dashed lines in the plots. a, c) Results from MD simulations on the open-state model with the SF initially configured to have only K+ ions (panel a) or alternating K+ / water molecules (panel c), respectively. b, d) Results from MD simulations on the inactivated model with the SF initially configured to have only K+ ions (panel b) or alternating K+ / water molecules (panel d), respectively.

Visualization of interactions for cationic astemizole (a), dofetilide (b), and quinidine (c) with different hERG channel models.

Each panel includes 3 subpanels showcasing drug interactions with the open-, inactivated, and closed-state hERG channel models. The estimated drug binding free energies, ΔGbind, are given in Rosetta energy units (R.E.U). In each subpanel, an overview of where the drug binds within the channel pore is shown on the upper left, a 3D visualization of interactions between each channel residue (blue, red, green, and tan residues are from the subunit A, B, C, or D, respectively) to the drug (magenta) is shown on the upper right, and a 2D ligand – protein interaction map is shown at the bottom. A continuous gray line depicts the contour of the protein binding site, and any breaks in this line indicate areas where the ligand is exposed to the solvent.

Data used for validating binding affinities from drug docking simulation with experiments

Correlation of predicted hERG drug binding affinities obtained from GALigandDock, in Rosetta Energy Units (R.E.U.), with experimental drug binding potencies (IC50) converted to kcal/mol.

Lower (more negative) binding energies indicate stronger binding. For each drug and hERG channel state, the top 50 out of 25,000 binding poses were clustered based on their root-mean-square deviations (RMSDs), and the average binding affinity from each cluster was used to represent the drug – ion channel interaction. a) A control scenario where only the open-state binding was considered, which is the conventional approach in ion channel pharmacology due to the lack of structural data for other states. b) A scenario that considers drug binding to different ion channel states (open, inactivated, and closed) predicted by our AlphaFold2 approach. The binding affinities for each hERG channel state were adjusted by the probability of that state occurring, based on a five-state model, and the drug’s ionization state at the experimental pH and temperature. This enabled a more accurate comparison between the computational drug binding affinities and experimental IC50 values, which were converted to kcal/mol. When accounting for drug binding to multiple channel states, the simulated binding affinities showed a 35.6% improvement in R² (0.61 vs. 0.45), a stronger correlation (Pearson’s r = 0.78 vs. 0.67), and greater statistical significance (P-value = 0.00001 vs. 0.00046) compared to the conventional approach of considering only open-state binding. This suggests that even a simplified multi-state model offers better predictive power, with further improvements likely when drug-induced gating modulation is included in future studies.

Transition rates in the hERG channel (IKr) Markov model

Voltage stimulation protocols and IC50 for drugs used in the model

Comparison of the SF in hERG closed- (a, d), open- (b, e), and inactivated-state (c, f) models. a, b,

c) Measurement of the distances between each carbonyl oxygen lining the conduction pathway in the SF. In the open- and closed-state models, S620 backbone carbonyl interacts with G626 and S624 backbone amide NH groups. In the inactivated-state model, the hydrogen bond between S620 and G626 is absent due to a reorientation of V625 backbone. However, at the bottom of the SF, S624 sidechain interacts with S623 backbone carbonyl from an adjacent subunit (denoted by *). d, e, f) View of the SF from the extracellular side. Large arrows indicate the rotation of the F627 side chains, while small arrows show the rotation of the loops that connect the upper SF to the S6 helix, all relative to the equivalent structural elements in the open-state model.

Interaction network analysis showcasing residue-residue interactions in the S5-P linker (I583 – Q592) and region surrounding the SF (S620 – N633).

a) An image of a hERG channel subunit with the analyzed S5-P linker and SF regions colored in light green and light blue, respectively. b) Heatmaps showing intrasubunit and intersubunit (marked by X) interactions between each residue in the analyzed regions. The interactions analyzed are hydrogen bonding, π stacking, cation-π, and salt bridges. Black cells indicate no interactions. Gray cells indicate an interaction is present in both states. Blue, orange, and green colored cells indicate the interaction is present only in the open, inactivated, or closed state, respectively, but not in the other state being compared in the map. White lines are added to separate S5-P linker residues from the SF region residues. c, d, e) Visualization of the interactions being present in one state but not the other. Gold-colored residues are involved in the interactions. Green-colored residues, named with an asterisk at the end, are from an adjacent chain but are interacting with gold-colored residues. Dashed lines represent hydrogen bonds.

Distance-based contact maps comparing intra- and intersubunit contacts between each model.

Two residues whose Cα atoms are within 6 Å of each other are considered to be in contact, provided there are no Cα atoms belonging to a third residue in between. Black cells indicate no contacts. Gray cells indicate a contact is present in both states being compared. Blue, orange, and green colored cells indicate the interaction is present only in the open, inactivated, or closed state, respectively, but not in the other state being compared in the map. Colored topology labels are included along the left and bottom edges of the maps showing the specific segments of the hERG channel to which the residues correspond.

Comparison of the S6 helix conformation for the hERG closed- (a), open- (b), and inactivated-state (c) models.

Residues E575 – L666 from the pore domain are visualized as dark gray ribbons. Selectivity filter (SF) residues and those on the S6 helix are shown with their backbone and side chains displayed as colored sticks. C atoms are gray, O are red, N are blue, S are yellow, H are not shown. The drug binding residues Y652 and F656 are highlighted in green.

Setup of MD simulations to assess ion conduction in the open and inactivated hERG channel models.

a) Initial configuration of the SF, set to fill with either all K+ ions (top), or alternating K+ and water molecules (bottom). b) An example MD simulation box showing a hERG channel model (shown in yellow surface representation) embedded in POPC lipid bilayer (shown as sticks) and solvated by an aqueous 0.3 M KCl solution (shown as a transparent surface with K+ and Cl- ions shown as purple and green balls, respectively).

Movement of K+ ions through hERG selectivity filter (SF).

The z coordinates of K+ ions are tracked as they traverse through the pore of the channel from the intracellular gate (lower y-axis limit) to the extracellular space (upper y-axis limit). Putative K+ binding sites in the SF (S0 – S5) are marked using blue dashed lines in the plots. a, c) Molecular dynamics (MD) simulations with the applied 500 mV membrane voltage of the open-state model with the SF initially configured to have only K+ ions (panel a) or alternating K+ / water molecules (panel c), respectively. b, d) MD simulations with the applied 500 mV membrane voltage of the inactivated-state model with the SF initially configured to have only K+ ions (panel b) or alternating K+ / water molecules (panel d), respectively. e, g) MD simulations without applied membrane voltage of the open-state model with the SF initially configured to have only K+ ions (panel e) or alternating K+ / water molecules (panel g), respectively. f, h) MD simulations without applied membrane voltage of the inactivated-state model with the SF initially configured to have only K+ ions (panel f) or alternating K+ / water molecules (panel h), respectively.

Analysis of modulations of the selectivity filter (SF) conformations and pore radii over the course of the 1 µs long molecular dynamics (MD) simulations.

The blue/orange-colored lines represent the average pore radii, and the shaded regions represent the standard deviation measured in MD simulations for a given Z value. The black lines represent the initial pore radii. The label on the left indicates the voltage of the MD simulations in each row.

Analysis of dynamics of the SF and pore conformations over the course of the 1 µs MD simulations.

a) Pore radius averaged over each 1 µs long MD simulations with (right) or without (left) applied membrane voltage. Open- and inactivated-state model MD simulations are notated as O and I, respectively, with the subscripts KK and WK denoting whether the SF initially configured to have only K+ ions or alternating K+ / water molecules, respectively. b) Ensembles of SF conformation over the course of each MD simulation superimposed. The golden-colored conformation indicates the initial conformation.

Cross-subunit distances between carbonyl oxygens of open-state hERG selectivity filter residues during MD simulations under different applied voltage and initial K+ ion position conditions.

Movement of potassium ions (denoted by differently colored lines) is shown at the bottom for reference. Red lines indicate initial distances. Labels 1 and 2 in red and blue, respectively, indicate a sequential dilation process exhibited by the hERG channel: the SF near residues F627 dilates first, followed by that around G628.

Sequential dilation steps of hERG upper selectivity filter (SF).

SF residues are shown as gray sticks, water molecules as red and white spheres, and K+ as purple spheres. The first step, occurring around 100 ns, involves the flipping of F627 carbonyl oxygen, creating a small dilation at this level. At 500 ns, further dilation can be seen at the level of residues F627 and G628 in one subunit. At 1000 ns, the entire upper region of the SF dilates further. Frames were taken from an MD simulation of the open-state hERG channel with K+ and water initially in the SF prior to transmembrane voltage of 750 mV being applied.

GALigandDock drug docking results to different hERG channel models.

Each bar plot represents the estimated free energy of binding in Rosetta energy units (R.E.U.) for the named drug to the open-, inactivated, and closed-state hERG channel models. Lower values mean more favorable binding. 25,000 docking poses were generated for each drug/channel model pairing. The top 50 poses were clustered, and the plotted energy represents the average free energy of the top cluster along with the standard deviation. The first suffixes (0), (+), and (±) indicate whether the drug is in the neutral, cationic, or zwitterionic form, respectively. The second suffixes * and † indicate validation from experimental studies (Alexandrou et al., 2006; Numaguchi et al., 2000; Perrin et al., 2008; Wang et al., 1997) showing whether the drug prefers binding to hERG inactivated state (*) or does not (†), respectively.