Oligomerization of the antimalarial drug target Pf ATP4 is essential for parasite survival

P-type ATPase (ATP4) is a Na efflux pump crucial for maintaining low [Na] in malaria parasites during their intraerythrocytic development cycle. In recent years, multiple studies have shown ATP4 to be the target of a large number of chemical scaffolds, including current candidate antimalarials KAE609 and SJ733. Here we show that ATP4 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 ATP4 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 ATP4. Wildtype and the mutated ATP4 genes were introduced at an ectopic locus in a line, in which endogenous ATP4 was conditionally expressed. Whereas the wildtype copy of ATP4 expressed from the ectopic locus was able to form the oligomeric complex, the mutant ATP4 failed to do so. Strikingly, unlike the wildtype, the mutant ATP4 failed to functionally complement the knockdown of the endogenous gene, leading to parasite demise. These results strongly suggest that co-translational oligomerization of ATP4 is essential for its function and for parasite survival.