Mutational Analysis of an Antimalarial Drug Target PfATP4

Among new antimalarials discovered over the past decade are multiple chemical scaffolds that target Plasmodium falciparum P-type ATPase (PfATP4). This essential protein is a Na+ pump responsible for the maintenance of Na+ homeostasis. PfATP4 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 PfATP4 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 PfATP4 critical for its function, as well as those that impart resistance to various PfATP4 inhibitors. To validate our model, we developed a genetic system involving merodiploid states of PfATP4 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 PfATP4. Phenotypic characterization of these mutants involved assessment of parasite growth, localization of mutated PfATP4, response to treatment with known PfATP4 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. Significance StatementPlasmodium falciparum ATP4 (PfATP4) is a Na+ efflux pump and represents an important target for antimalarial drugs with nanomolar potency. However, the structure of PfATP4 is unknown, prompting the development of new methodologies to investigate the structure/function relationship. Here, we introduce a dynamic homology modeling approach to (a) identify key residues essential for PfATP4 function and (b) provide a structural basis to understand resistance-associated mutations. To validate these predictions, we developed a genetic system to manipulate the PfATP4 gene to assess the phenotypic consequences of such changes. Our results support the utility of combining homology modeling and genetics to gain functional insights into an antimalarial drug target.