Structural Bases for the Unconventional Activity of a Viroporin Channel

1. Pore-forming activity of p7 protein experimentally measured in surrogates of the ER membrane. (a) ER-GUV distribution according to the percentage of permeabilization per vesicle measured against solutes of increasing sizes. Samples were treated with p7 (0.2 μM), and percentages of entry into single vesicles calculated for the different solutes after 30 min incubation. Confocal micrographs on top depict selected vesicles comparing degrees of permeabilization to Alexa Fluor 488 and dextrans with increasing MWs. Fluorescence of GUV membranes and solutes are colored in orange and green, respectively. Note that the lipid bilayer integrity is retained in vesicles fully permeabilized to Alexa Fluor or 4 kDa dextrans. Scale bars, 10 μm. (b) Pore size (radius) as a function of the two coexisting ion conductance levels described in ref . Radii were estimated from main current jump intensities and applied voltage assuming a cylindrical geometry for the permeating structure (for a detailed description of the procedure followed, please see also ref ). The permeability levels to solutes of increasing sizes determined in the previous panel are plotted in parallel for comparison. The shadowed…

2. Stability and pore-forming activity of the TMH1 hexamer model in the ER-LB system with NaCl. (a) Helix-helix packing interactions observed between consecutive h2–h3 hairpins at the end of the double-hairpin hexamer/ER-LB system MD simulations (Figure S1). The right panel illustrates the inclination angle between interacting helices 2 and 3 of adjacent hairpins. (b) TMH1 model generation and overall structure. (c) Top and side views of the TMH1 hexamer inserted into the ER-LB. Snapshots were selected after equilibration (left) and after running the simulation (right). (d) RMSD changes during the simulations. Changes over time are shown for the membrane-inserted protein and its different components, i.e., s-h1, the h2–h3 hairpin, and h4, as indicated in the panels (see Figure S1a). (e) HOLE-calculated, time-averaged pore radius profiles in three different simulations. Blue, red, and orange dashed lines indicate the radii of water and hydrated Na+ and K+, respectively. The black dotted line marks a 1 nm diameter pore.

3. Stability and pore-forming activity of the TMH26 hexamer model in the ER-LB system. (a) TMH26 hexamer model calculated by ColabFold v1.5.5 using the hexamer of extended monomers TMH1 as the starting structure. Blue and green colors denote the high pLDDT of the model (see Figure S4 for a comparison with a low pLDDT model). (b) Top and side views of the TMH2 hexamer inserted into the ER-LB. Snapshots were selected after equilibration (left) and at the end of the simulation (right). (c) RMSD changes occurring during the simulations in the full protein and its different sections. The traces in black, red, and blue correspond to three different replicas. (d) HOLE-calculated, time-averaged pore radius profiles in three different simulations. Blue, orange, and red dashed lines indicate the radii of water, hydrated K+, and Na+, respectively. The black dotted line marks a 1 nm diameter pore.

4. Stability and pore-forming activities of TMH27 in the ER-LB system. (a) TMH27 heptamer model calculated by ColabFold v1.6.1 using TMH1 as the template structure. Blue and green colors denote the high pLDDT of the model. (b) Top and side views of the TMH2 heptamer inserted into the ER-LB. Snapshots were selected after equilibration (left) and at the end of the simulation (right). (c) RMSD changes occurring during the simulations in the full protein and its different sections. The traces in black, red, and blue correspond to three different replicas. (d) HOLE-calculated, time-averaged pore radius profiles in three different simulations. Blue, orange, and red dashed lines indicate the radii of water, hydrated K+, and Na+, respectively. The black dotted line marks a 1 nm diameter pore.

5. Sodium transport through the predicted heptamer is associated with Glu21 and the hydrophobic residues Ile20, Leu24, and Leu28. Blue dotted lines represent the average positions of the lipid phosphate groups in the extracellular (EC) and intracellular (IC) membrane leaflets. The black arrow indicates the time at which the snapshot was taken. During the 2.5 ns traversal of the sodium ion from the EC to the IC side, the ion was coordinated by Glu21 and subsequently interacted with Ile20, Leu24, and Leu28, while remaining coordinated mainly by water throughout the process.

6. Stability and pore-forming activities of TMH25 in the ER-LB system. (a) TMH25 model calculated by ColabFold v1.6.1 using TMH1 as the template structure. Blue and green colors denote the high pLDDT of the model. (b) Top and side views of the TMH25 pentamer inserted into the ER-LB. Snapshots were selected after equilibration (left) and at the end of the simulation (right). (c) RMSD changes occurring during the simulations in the full protein and its different sections. The traces in black, red, and blue correspond to three different replicas. (d) HOLE-calculated, time-averaged pore radius profiles in three different simulations. Blue, orange, and red dashed lines indicate the radii of water, hydrated K+, and Na+, respectively. The black dotted line marks a 1 nm diameter pore.

7. Pore surface diagrams and evolution of the radii over simulation times (left and right panels, respectively). Pore radius profiles correspond to the minimum radius along the pore axis, as calculated using HOLE. Individual replicas are shown in different colors for each system. Replicates extending to 2 μs were truncated to 0.5 μs to match the simulation time of the other replicas.

8. Interactions of channel blockers with TMH1 and TMH26 hexamers in membranes. Docking results for (a) verapamil and (b) amantadine are shown at the start of the simulation and after 200 ns for the complexes in the ER-LB. Each molecule is depicted in a distinct color, while the protein is shown in gray using a cartoon representation, viewed perpendicular to the membrane plane.