Structural Bases for the Unconventional Activity of a Viroporin Channel.

Viroporins alter the permeability of cell membranes and regulate the initiation/progression of the viral infection cycle. However, the "unconventional" membrane channel behavior displayed by many members of the family challenges their general validation as therapeutic targets. The reported capacity of the Classical Swine Fever Virus p7 viroporin for establishing ion-conducting channels of different sizes exemplifies that behavior. Using all-atom molecular dynamics (MD) simulations, we attempted to elucidate the structural basis and mechanisms underlying the atypical activity of p7. Based on AlphaFold-predicted CSFV p7 hexamer structures with folded-back helical hairpins, we first generated monomers that spanned the entire thickness of the lipid bilayer. Next, we assembled oligomers of varying stoichiometry (pentamers, hexamers, and heptamers) based on those extended transmembrane hairpin (TMH) protomers. We focused on two hexameric models: TMH1, preserving the helix-helix packing interactions observed in the initial model, and TMH26, generated using TMH1 as a template for ColabFold. In line with experimental evidence, the simulations revealed that both structural architecture and oligomeric state determine pore organization in ER-like membranes. TMH1 hexamers and TMH27 heptamers adopted wide pore geometries with extensive hydration and ion accessibility, whereas TMH26 hexamers sampled narrower, more compact hydrated pore states. TMH25 pentamers remained predominantly nonconductive. Dynamic interactions between transmembrane helices and the number of protomers incorporated into the membrane-embedded structure appeared to be instrumental for this capacity. These findings reveal how variation in oligomeric state and helix packing can generate pores with different dimensions and hydration properties, providing a structural framework for interpreting p7's experimentally observed capacity to induce multiple conductance states and size-selective membrane permeabilization.