PfATP4 models

Among new antimalarials discovered over the past decade are multiple chemical scaffolds that target <i>Plasmodium falciparum</i> P-type ATPase (<i>Pf</i>ATP4). This essential protein is a Na⁺ pump responsible for the maintenance of Na⁺ homeostasis. <i>Pf</i>ATP4 belongs to the type 2D subfamily of P-type ATPases, for which no structures have been determined. To gain better insight into the structure/function relationship of this validated drug target, we generated a homology model of <i>Pf</i>ATP4 based on SERCA, a P2A-type ATPase, and refined the model using molecular dynamics in its explicit membrane environment. This model predicted several residues in <i>Pf</i>ATP4 critical for its function, as well as those that impart resistance to various <i>Pf</i>ATP4 inhibitors. To validate our model, we developed a genetic system involving merodiploid states of <i>Pf</i>ATP4 in which the endogenous gene was conditionally expressed, and the second allele was mutated to assess its effect on the parasite. Our model predicted residues involved in Na⁺ coordination as well as the phosphorylation cycle of <i>Pf</i>ATP4. Phenotypic characterization of these mutants involved assessment of parasite growth, localization of mutated <i>Pf</i>ATP4, response to treatment with known <i>Pf</i>ATP4 inhibitors, and evaluation of the downstream consequences of Na⁺ influx. Our results were consistent with modeled predictions of the essentiality of the critical residues. Additionally, our approach confirmed the phenotypic consequences of resistance-associated mutations as well as a potential structural basis for the fitness cost associated with some mutations. Taken together, our approach provides a means to explore the structure/function relationship of essential genes in haploid organisms.