A Soluble Expression Construct of the Isolated Catalytic Domain of Plasmodium falciparum ATP4 Exhibits ATPase Activity Independent of a γ-Phosphate Receiving Aspartate.

The sodium/proton-exchanging ATPase of Plasmodium falciparum malaria parasites, PfATP4, is an emerging drug target. Inhibition results in detrimental cell swelling due to cytosolic accumulation of sodium and alkalization. PfATP4 is a sodium-releasing type II P-type ATPase restricted to apicomplexan parasites. Experimental data on structure-function relationships of the isolated protein are absent. Here, we produced and purified the soluble catalytic domain of PfATP4 and evaluated kinetic properties by in vitro phosphate colorimetry. The protein exhibited Mg2+-dependent ATPase activity at the same order of magnitude as the native cellular PfATP4 and was insensitive to the presence of sodium. AlphaFold 3-based structure and ATP/Mg2+ interaction predictions identified key residues of the nucleotide binding domain (Lys619, Lys652, Arg703). Replacement of the lysines by methionine decreased the enzymatic activity to one quarter. Individual mutation of the putative Mg2+-coordinating Asp865 of the phosphorylation domain was tolerated, while a joint replacement with Asp869 decreased ATPase again to one quarter. Mutation of the putative γ-phosphate receiving Asp451 maintained the rate of Pi release. Our data attribute typical functional roles for P-type ATPases to the basic and acidic residues of the soluble PfATP4 catalytic domain and show that its ATP hydrolysis is independent of phosphorylation of Asp451.