Deciphering Membrane Pore Formation Mechanisms of Plasmodium falciparum Perforin-Like Protein 1 (PfPLP1).

Membrane Attack Complex/Perforin (MACPF) domain proteins are β-pore forming toxins (β-PFTs) involved in the pathogenesis of various organisms. Among them, Perforin-like proteins (PLPs), produced by Plasmodium species, play essential roles in parasite invasion and egress. Due to increasing drug resistance in Plasmodium, PLPs represent promising but underexplored therapeutic targets, largely due to the lack of structural and mechanistic data. This study investigates the binding and pore formation mechanism of the Plasmodium falciparum PLP1 (PfPLP1), which is expressed during the human life cycle of the parasite. We modeled PfPLP1 structure and performed both all-atom and coarse-grained molecular dynamics simulations in soluble and membrane-associated states. PfPLP1 comprises two domains, a canonical MACPF domain and a β-pleated sheet domain- apicomplexan perforin β-domain (APCβ). Initial membrane binding is mediated by cationic residues at the base of the APCβ domain, which interact with the polar headgroups of the lipids from the host cell membrane. We analyzed the membrane-inserted tetrameric form where water molecules were observed to penetrate between the tetramer and the lipid bilayer, initiating pore opening. During this process, lipids reorganize into a toroidal edge to shield their hydrophobic tails, while water mixes with lipid headgroups in a disordered, heterogeneous fashion. Larger oligomeric assemblies show lateral displacement of lipids and a clear tendency to form pore-like structures. This study provides molecular insights into PfPLP1's membrane binding and pore-forming behavior in both monomeric and oligomeric forms. The outcome of this study would be applicable in understanding pore formation mechanism in other PLPs and similar toxins.