Oligomerization of the antimalarial drug target PfATP4 is essential for parasite survival

Abstract

AbstractPlasmodium falciparum P-type ATPase (PfATP4) is a Na+ efflux pump crucial for maintaining low [Na+]i in malaria parasites during their intraerythrocytic development cycle. In recent years, multiple studies have shown PfATP4 to be the target of a large number of chemical scaffolds, including current candidate antimalarials KAE609 and SJ733. Here we show that PfATP4 exists as a large complex. Immunopurification and proteomic studies revealed the complex to be homooligomeric in nature. The complex appears to be assembled co-translationally. Phylogenetic analysis suggests that ATP4 from apicomplexans and chromerids form a distinct class of P-type ATPases having fewer transmembrane helices compared to their orthologues. We hypothesized that reduction of transmembrane helices in PfATP4 might necessitate oligomerization to maintain its function. We further suspected potential involvement of π-π interactions between aromatic amino acids within the terminal transmembrane helix of each monomer to be critical for oligomerization. To test this hypothesis, we mutated three aromatic amino acids in the last transmembrane helix of PfATP4. Wildtype and the mutated PfATP4 genes were introduced at an ectopic locus in a P. falciparum line, in which endogenous PfATP4 was conditionally expressed. Whereas the wildtype copy of PfATP4 expressed from the ectopic locus was able to form the oligomeric complex, the mutant PfATP4 failed to do so. Strikingly, unlike the wildtype, the mutant PfATP4 failed to functionally complement the knockdown of the endogenous gene, leading to parasite demise. These results strongly suggest that co-translational oligomerization of PfATP4 is essential for its function and for parasite survival.Significance StatementPlasmodium falciparum ATP4 (PfATP4) is a Na+ efflux pump and is the target of at least two antimalarial drug candidates (KAE609, SJ733) currently in clinical trials. With a rapid parasite clearance rate (t99=12h) in initial clinical studies, PfATP4-active drugs present the prospect of taking us a step closer to malaria elimination in a world that is currently threatened by artemisinin resistance. In this study, we have established that PfATP4 exists as a homo-oligomeric complex, which is assembled co-translationally through interactions facilitated by aromatic amino acids in its last transmembrane helix. Crucially, its existence as a complex is essential for its function. This exposes a vulnerability in the target that could be potentially exploited for drug design.