Michael W Mather

The Plasmodium falciparum sodium efflux pump Pf ATP4 is a leading antimalarial target, but suffers from a lack of high-resolution structural information needed to identify functionally important features in conserved regions and guide rational design of next generation inhibitors. Here, we determine a 3.7Å cryoEM structure of Pf ATP4 purified from CRISPR-engineered P. falciparum parasites, revealing a previously unknown, apicomplexan-specific binding partner, Pf ABP, which forms a conserved, likely modulatory interaction with Pf ATP4. The discovery of Pf ABP presents a new avenue for designing novel Pf ATP4 inhibitors.The Plasmodium falciparum sodium efflux pump Pf ATP4 is a leading antimalarial target, but suffers from a lack of high-resolution structural information needed to identify functionally important features in conserved regions and guide rational design of next generation inhibitors. Here, we determine a 3.7Å cryoEM structure of Pf ATP4 purified from CRISPR-engineered P. falciparum parasites, revealing a previously unknown, apicomplexan-specific binding partner, Pf ABP, which forms a conserved, likely modulatory interaction with Pf ATP4. The discovery of Pf ABP presents a new avenue for designing novel Pf ATP4 inhibitors.

Plasmodium 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.

Abstract The battle against malaria has been substantially impeded by the recurrence of drug resistance in Plasmodium falciparum , the deadliest human malaria parasite. To counter the problem, novel antimalarial drugs are urgently needed, especially those that target unique pathways of the parasite, since they are less likely to have side effects. The mitochondrial type II NADH dehydrogenase of P. falciparum , PfNDH2 (PF3D7_0915000), has been considered a good prospective antimalarial drug target for over a decade, since malaria parasites lack the conventional multi-subunit NADH dehydrogenase, or Complex I, present in the mammalian mitochondrial electron transport chain (mtETC). Instead, Plasmodium parasites contain a single subunit NDH2, which lacks proton pumping activity and is absent in humans. A significant amount of effort has been expended to develop PfNDH2 specific inhibitors, yet the essentiality of PfNDH2 has not been convincingly verified. Herein, we knocked out PfNDH2 in P. falciparum via a CRISPR/Cas9 mediated approach. Deletion of PfNDH2 does not alter the parasite’s susceptibility to multiple mtETC inhibitors, including atovaquone and ELQ-300. We also show that the antimalarial activity of the fungal NDH2 inhibitor HDQ and its new derivative CK-2-68 is due to inhibition of the parasite cytochrome bc 1 complex rather than PfNDH2. These compounds directly inhibit the ubiquinol-cytochrome c reductase activity of the malarial bc 1 complex. Our results call into question the validity of PfNDH2 as an antimalarial drug target. Importance For a long time, PfNDH2 has been considered an attractive antimalarial drug target. However, the conclusion that PfNDH2 is essential was based on preliminary and incomplete data. Here we generate a PfNDH2 KO (knockout) parasite in the blood stages of Plasmodium falciparum , showing that the gene is not essential. We also show that previously reported PfNDH2-specific inhibitors kill the parasites primarily via targeting the cytochrome bc 1 complex, not PfNDH2. Overall, we provide genetic and biochemical data that help to resolve a long-debated issue in the field regarding the potential of PfNDH2 as an antimalarial drug target.