Affinage · Plasmodium

ATP4

PF3D7_1211900 · non-SERCA-type Ca2+ -transporting P-ATPase

Annotated
2026-09-08
53 papers in source corpus 15 papers cited in narrative 15 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

PF3D7_1211900 (ATP4) is an essential P-type cation ATPase of the intraerythrocytic P. falciparum plasma membrane that maintains parasite ion homeostasis by extruding Na+ against an inward gradient to keep cytosolic [Na+] low, and it co-regulates cytosolic pH (PMID:23414762, PMID:25145582, PMID:39773028). The protein is a ~190 kDa P-type ATPase expressed throughout the asexual erythrocytic stages and in free merozoites, concentrated at the parasite periphery/plasma membrane (PMID:8813672, PMID:39773028). Its catalytic mechanism has been dissected biochemically: the isolated soluble catalytic domain shows Mg2+-dependent ATPase activity that depends on nucleotide-binding residues (Lys619, Lys652, Arg703) and Mg2+-coordinating residues (Asp865/Asp869), and homology modelling plus a conditional-allele genetic system have defined residues critical for Na+ coordination and the phosphorylation cycle whose mutation impairs both growth and Na+ regulation (PMID:40091859, PMID:39773028). A 3.7 Å cryo-EM structure of the endogenous protein revealed an apicomplexan-specific binding partner, PfABP (PMID:41115914). ATP4 is the molecular target of multiple structurally distinct antimalarial chemotypes — spiroindolones/cipargamin, pyrazoleamides, aminopyrazoles, dihydroisoquinolones such as (+)-SJ733, and others — whose inhibition disrupts Na+ homeostasis and triggers downstream eryptotic changes (increased membrane rigidity, phosphatidylserine externalization) leading to parasite death, and several of these compounds also block transmission to mosquitoes (PMID:20813948, PMID:23414762, PMID:25453091, PMID:25422853, PMID:25322084, PMID:25145582, PMID:40228810). Resistance-conferring mutations in the pfatp4 gene, including the clinically relevant G358S allele that lowers Na+ affinity, reduce inhibitor sensitivity, confirming ATP4 as the on-target of these compounds (PMID:28542423, PMID:36180431, PMID:41308470).

Mechanistic history

Synthesis pass · year-by-year structured walk · 8 steps
  1. 1996 Medium

    Establishing that the gene product is a large P-type ATPase present at the parasite periphery throughout asexual development set the stage for a membrane-transport function.

    Evidence Gene cloning with Western blot and immunofluorescence in P. falciparum asexual stages and merozoites

    PMID:8813672

    Open questions at the time
    • Localization described as peripheral compartments, not yet resolved as plasma membrane
    • No transported ion identified
    • No functional transport assay
  2. 2001 Medium

    Heterologous expression provided the first direct enzymatic readout, showing a vanadate- and cyclopiazonic-acid-sensitive but ouabain-/thapsigargin-resistant ATPase, distinguishing it pharmacologically from SERCA and Na/K-ATPases.

    Evidence Xenopus oocyte expression with pharmacological ATPase assays and stage-specific qPCR in P. falciparum

    PMID:11145964

    Open questions at the time
    • Ca2+-stimulation reported here was later reframed by Na+-pump studies
    • Single-lab heterologous system
    • Physiological transported ion not resolved
  3. 2010 High

    Resistance selection against spiroindolones mapped mutations to pfatp4, identifying the gene as an antimalarial drug target and motivating direct interrogation of its function.

    Evidence Resistance selection with whole-genome sequencing and protein-synthesis inhibition assays in P. falciparum

    PMID:20813948

    Open questions at the time
    • Physiological pump activity not yet defined
    • Downstream mechanism linking inhibition to death unresolved
  4. 2013 High

    Live-cell ion measurements resolved the transported species, defining the protein as a plasma-membrane Na+ efflux ATPase and explaining how its inhibition perturbs cytosolic Na+.

    Evidence Fluorescent Na+ indicator measurements with genetic resistance-mutation epistasis in P. falciparum

    PMID:23414762

    Open questions at the time
    • Mechanism of H+ counter-transport not directly demonstrated
    • Structural basis of Na+ coordination unresolved
  5. 2014 High

    Multiple chemotypes and phenotypic studies established a unified mechanism-of-action: ATP4 inhibition disrupts Na+ (and pH) homeostasis, drives eryptotic changes and death, and blocks transmission, while defining the fitness cost of resistance.

    Evidence Resistance mapping, ion/pH homeostasis and membrane-biophysics assays, transmission assays, and in vivo models for pyrazoleamides, aminopyrazoles, SJ733, and Malaria Box compounds in P. falciparum

    PMID:25145582 PMID:25322084 PMID:25422853 PMID:25453091

    Open questions at the time
    • Whether pH dysregulation is a direct pump activity or downstream consequence not resolved
    • Molecular details of inhibitor binding site not defined
  6. 2017 High

    CRISPR introduction of specific point mutations proved the causal genetic link between pfatp4 alleles and SJ733 resistance, moving beyond correlative resistance mapping.

    Evidence Plasmid-free CRISPR/Cas9 with ssODN editing and growth-inhibition assays in P. falciparum

    PMID:28542423

    Open questions at the time
    • Structural interpretation of the mutated residues not provided
  7. 2022 High

    The clinically relevant G358S mutation linked field/clinical resistance to a measurable reduction in Na+ affinity without a growth or transmission cost, and its conservation in Toxoplasma gondii extended the mechanism across apicomplexans.

    Evidence Clinical resistance marker sequencing, ion homeostasis assays, and cross-species genetic introduction into T. gondii ATP4

    PMID:36180431

    Open questions at the time
    • Structural basis of how G358S alters Na+ and inhibitor binding not shown here
  8. 2025 High

    Structure, biochemistry, and residue-level genetics converged to define the catalytic machinery, plasma-membrane localization, Na+-coordination and phosphorylation-cycle residues, a novel binding partner PfABP, and direct drug engagement.

    Evidence 3.7 Å cryo-EM of endogenous protein, recombinant catalytic-domain ATPase assays with mutagenesis, homology modelling plus conditional-allele genetics, SPP target engagement, and N-acetamide indole in vitro Na+-ATPase inhibition in P. falciparum

    PMID:39773028 PMID:40091859 PMID:40228810 PMID:41115914 PMID:41308470

    Open questions at the time
    • Functional role of PfABP not experimentally defined
    • Catalytic-domain assay lacks full-length structural validation
    • Asp451-independent Pi release complicates a simple phosphoenzyme model

Open questions

Synthesis pass · forward-looking unresolved questions
  • The molecular role of the PfABP binding partner, the precise inhibitor-binding site, and the mechanistic coupling of Na+ efflux to cytosolic pH regulation remain open.
  • No functional assay defining what PfABP contributes to pump activity
  • Inhibitor binding pocket not resolved structurally in a drug-bound complex
  • Direct demonstration of counter-ion (H+) transport still lacking

Mechanism profile

Synthesis pass · controlled-vocabulary classification
Partners
PFABP (APICOMPLEXAN-SPECIFIC BINDING PARTNER) [#10]
Complex memberships
PfATP4–PfABP complex (cryo-EM, apicomplexan-specific binding partner) [#10]

Evidence

Reading pass · 15 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2001 PfATP4 (PF3D7_1211900) expressed in Xenopus laevis oocytes exhibits Ca2+-dependent ATPase activity stimulated over a broad [Ca2+]free range (3.2–320 µM), is resistant to inhibition by ouabain (200 µM) or thapsigargin (0.8 µM), but is inhibited by vanadate (1 mM) or cyclopiazonic acid (1 µM). mRNA expression is variable across asexual stages with a maximal ~5-fold increase in meronts compared with rings. Xenopus oocyte expression, ATPase activity assays with pharmacological inhibitors, quantitative PCR on synchronized asexual stages The Journal of biological chemistry Medium 11145964
1996 The PfATP4 (PF3D7_1211900) gene encodes a ~190 kDa P-type ATPase protein that is detectable immunologically throughout the asexual erythrocytic stages and in free merozoites. Immunofluorescence microscopy shows the protein is concentrated in discrete compartments at the periphery of the parasite. An 8 kb transcript appears shortly after erythrocyte invasion but is not detected in later stages, while the protein persists. Gene cloning, immunological detection (Western blot), immunofluorescence microscopy Molecular and biochemical parasitology Medium 8813672
2010 Spiroindolone antimalarials (including NITD609) rapidly inhibit protein synthesis in P. falciparum, and this effect is ablated in parasites bearing nonsynonymous mutations in the gene encoding PfATP4 (PF3D7_1211900), establishing PfATP4 as the target of spiroindolones. Resistance selection with whole-genome sequencing identifying mutations in PfATP4; protein synthesis inhibition assay Science (New York, N.Y.) High 20813948
2013 PfATP4 (PF3D7_1211900) localizes to the parasite plasma membrane and functions as a Na+ efflux ATPase, actively extruding Na+ against an inward gradient to maintain low cytosolic [Na+]. Spiroindolones cause a profound disruption of Na+ homeostasis in P. falciparum that is attenuated in parasites bearing resistance-conferring mutations in PfATP4; those mutant parasites also show some impairment of Na+ regulation. Fluorescent Na+ indicator measurements in live parasites; genetic resistance-conferring mutations in PfATP4 characterized by whole-genome sequencing; ion homeostasis assays Cell host & microbe High 23414762
2014 Treatment of P. falciparum-infected erythrocytes with (+)-SJ733, which acts on PfATP4 (PF3D7_1211900), causes rapid perturbation of parasite Na+ homeostasis followed by increased membrane rigidity and externalization of phosphatidylserine in infected cells, consistent with eryptosis/senescence. Resistance-conferring mutations in pfatp4 carry a high fitness cost. Ion homeostasis assays (fluorescent Na+ indicators), membrane rigidity measurements, phosphatidylserine externalization assay, in vivo mouse model, resistance selection with whole-genome sequencing Proceedings of the National Academy of Sciences of the United States of America High 25453091
2014 Pyrazoleamide antimalarials disrupt Na+ regulation in blood-stage P. falciparum in a manner dependent on PfATP4 (PF3D7_1211900); mutations in PfATP4 (in combination with mutations in PfCDPK5) are necessary to confer full pyrazoleamide resistance, establishing PfATP4 as part of their mechanism of action. Resistance selection, whole-genome sequencing, gene transfer (complementation), Na+ homeostasis assay Nature communications High 25422853
2014 Aminopyrazole antimalarials (e.g. GNF-Pf4492) target PfATP4 (PF3D7_1211900): three independently derived resistant lines each acquired independent mutations in PfATP4. The aminopyrazoles disrupt intracellular Na+ homeostasis and block parasite transmission to mosquitoes, phenocopying spiroindolone effects. Resistance selection, whole-genome sequencing of three independent resistant lines, Na+ homeostasis assay, gametocyte/transmission assay ACS chemical biology High 25322084
2014 Twenty-eight compounds from the Malaria Box affect P. falciparum Na+ and pH regulation consistent with PfATP4 (PF3D7_1211900) inhibition; six chemically diverse compounds showed reduced antiplasmodial activity against spiroindolone-resistant parasites carrying pfatp4 mutations, and resistance selection with two compounds yielded parasites with new mutations in pfatp4. This establishes PfATP4 as a target for diverse chemotypes and indicates it co-regulates both Na+ and cytosolic pH (alkalinization). Ion homeostasis screening (fluorescent Na+ and pH indicators), cross-resistance testing against pfatp4 mutant parasites, in vitro resistance selection with whole-genome sequencing Molecular microbiology High 25145582
2017 CRISPR/Cas9-mediated introduction of specific point mutations into pfatp4 (PF3D7_1211900) using a plasmid-free ribonucleoprotein + ssODN approach confirmed that those mutations confer resistance to the antimalarial SJ733, validating the genetic link between PfATP4 and SJ733 action. Plasmid-free CRISPR/Cas9 genome editing, ssODN repair template, whole-genome sequencing, growth inhibition assays PloS one High 28542423
2022 A G358S mutation in PfATP4 (PF3D7_1211900) confers clinically relevant resistance to cipargamin and (+)-SJ733, allowing parasites to withstand micromolar concentrations while remaining susceptible to non-PfATP4-targeting antimalarials. G358S reduces the affinity of PfATP4 for Na+ and is associated with increased resting cytosolic [Na+], but causes no detectable defect in parasite growth or transmissibility. The equivalent G358S mutation in Toxoplasma gondii ATP4 also decreases sensitivity to cipargamin and SJ733. Clinical trial resistance marker sequencing; functional ion homeostasis assays; genetic introduction of G358S into Toxoplasma gondii ATP4; parasite growth and transmissibility assays Nature communications High 36180431
2025 A 3.7 Å cryo-EM structure of PfATP4 (PF3D7_1211900) purified from CRISPR-engineered P. falciparum parasites reveals a previously unknown apicomplexan-specific binding partner, PfABP, which forms a conserved interaction with PfATP4 and likely plays a modulatory role. CRISPR engineering, native protein purification, cryo-EM structure determination at 3.7 Å Nature communications High 41115914
2025 The isolated soluble catalytic domain of PfATP4 (PF3D7_1211900) exhibits Mg2+-dependent ATPase activity in vitro at rates comparable to native cellular PfATP4. Mutation of key nucleotide-binding residues (Lys619, Lys652, Arg703 → Met) decreases enzymatic activity to ~one quarter. Mutation of Mg2+-coordinating residues Asp865+Asp869 together also reduces activity to ~one quarter. Mutation of the putative γ-phosphate-receiving Asp451 does not prevent Pi release, indicating ATP hydrolysis by this domain proceeds independently of phosphorylation at Asp451. Recombinant protein expression and purification, in vitro phosphate colorimetric ATPase assay, site-directed mutagenesis, AlphaFold 3 structural modelling Molecular microbiology Medium 40091859
2025 Homology modelling of PfATP4 (PF3D7_1211900) combined with a merodiploid conditional expression genetic system identified residues critical for Na+ coordination and the phosphorylation cycle. Phenotypic characterization of mutants confirmed that PfATP4 is localized to the parasite plasma membrane, that mutation of these residues impairs parasite growth and Na+ regulation, and that resistance-associated mutations reduce inhibitor sensitivity with some conferring a fitness cost consistent with model predictions. Homology modelling (SERCA-based), molecular dynamics simulation, CRISPR-mediated merodiploid conditional allele system, parasite growth assays, subcellular localization (imaging), Na+ homeostasis assays Proceedings of the National Academy of Sciences of the United States of America High 39773028
2025 Solvent-induced proteome profiling (SPP) identified PfATP4 (PF3D7_1211900; annotated as non-SERCA-type Ca2+-transporting P-ATPase) as the protein target of cipargamin in P. falciparum lysates, demonstrating ligand-induced stabilization of PfATP4 consistent with direct drug-protein interaction. Solvent proteome profiling (SPP), mass spectrometry-based proteomics International journal for parasitology. Drugs and drug resistance Medium 41308470
2025 N-acetamide indole compounds inhibit PfATP4 (PF3D7_1211900): resistance selection and whole-genome sequencing revealed mutations in PfATP4; analogs showed reduced potency against PfATP4 resistance-mutant parasites; metabolomic signature matched that of the PfATP4 inhibitor KAE609; and in vitro inhibition of Na+-dependent ATPase activity was demonstrated. The compounds also inhibit gamete development and block transmission to mosquitoes. Resistance selection, whole-genome sequencing, cross-resistance assays, metabolomics, Na+-dependent ATPase inhibition assay in vitro, gametocyte/transmission assay, P. berghei mouse model Journal of medicinal chemistry High 40228810

Source papers

Stage 0 corpus · 53 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2009 Artemisinin resistance in Plasmodium falciparum malaria. The New England journal of medicine 2627 19641202
2010 Spiroindolones, a potent compound class for the treatment of malaria. Science (New York, N.Y.) 772 20813948
2003 Artemisinins target the SERCA of Plasmodium falciparum. Nature 720 12931192
2005 Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA-type PfATPase6. Lancet (London, England) 367 16325698
2006 Current perspectives on the mechanism of action of artemisinins. International journal for parasitology 211 17005183
2014 (+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium. Proceedings of the National Academy of Sciences of the United States of America 199 25453091
2005 A single amino acid residue can determine the sensitivity of SERCAs to artemisinins. Nature structural & molecular biology 192 15937493
2013 Na(+) regulation in the malaria parasite Plasmodium falciparum involves the cation ATPase PfATP4 and is a target of the spiroindolone antimalarials. Cell host & microbe 187 23414762
2005 The 'permeome' of the malaria parasite: an overview of the membrane transport proteins of Plasmodium falciparum. Genome biology 146 15774027
2006 Real-time quantitative PCR with SYBR Green I detection for estimating copy numbers of nine drug resistance candidate genes in Plasmodium falciparum. Malaria journal 142 16420686
1982 Calcium transport of Plasmodium chabaudi-infected erythrocytes. The Journal of cell biology 123 6288729
2003 Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem. The Journal of cell biology 118 12682086
2014 Pyrazoleamide compounds are potent antimalarials that target Na+ homeostasis in intraerythrocytic Plasmodium falciparum. Nature communications 104 25422853
1997 The complete inventory of the yeast Saccharomyces cerevisiae P-type transport ATPases. FEBS letters 103 9224683
2016 A broad analysis of resistance development in the malaria parasite. Nature communications 102 27301419
2015 The malaria parasite cation ATPase PfATP4 and its role in the mechanism of action of a new arsenal of antimalarial drugs. International journal for parasitology. Drugs and drug resistance 90 26401486
2001 Expression and functional characterization of a Plasmodium falciparum Ca2+-ATPase (PfATP4) belonging to a subclass unique to apicomplexan organisms. The Journal of biological chemistry 84 11145964
2014 Diverse chemotypes disrupt ion homeostasis in the Malaria parasite. Molecular microbiology 77 25145582
2014 Mutations in the P-type cation-transporter ATPase 4, PfATP4, mediate resistance to both aminopyrazole and spiroindolone antimalarials. ACS chemical biology 76 25322084
2003 The digestive food vacuole of the malaria parasite is a dynamic intracellular Ca2+ store. The Journal of biological chemistry 72 12740366
1993 Cloning of a Ca(2+)-ATPase gene of Plasmodium falciparum and comparison with vertebrate Ca(2+)-ATPases. Journal of cell science 71 8314897
1998 Inositol 1,4,5-trisphosphate induced Ca2+ release from chloroquine-sensitive and -insensitive intracellular stores in the intraerythrocytic stage of the malaria parasite P. chabaudi. Biochemical and biophysical research communications 68 9535800
2008 Plasmodium in the postgenomic era: new insights into the molecular cell biology of malaria parasites. International review of cell and molecular biology 61 18544493
2006 Antimalarial drugs: recent advances in molecular determinants of resistance and their clinical significance. Cellular and molecular life sciences : CMLS 61 16699808
2020 Safety, tolerability, pharmacokinetics, and antimalarial efficacy of a novel Plasmodium falciparum ATP4 inhibitor SJ733: a first-in-human and induced blood-stage malaria phase 1a/b trial. The Lancet. Infectious diseases 57 32275867
2022 Efficacy of Cipargamin (KAE609) in a Randomized, Phase II Dose-Escalation Study in Adults in Sub-Saharan Africa With Uncomplicated Plasmodium falciparum Malaria. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 56 34410358
2011 The Plasmodium falciparum Ca(2+)-ATPase PfATP6: insensitive to artemisinin, but a potential drug target. Biochemical Society transactions 55 21599655
1999 Calcium homeostasis and signaling in the blood-stage malaria parasite. Parasitology today (Personal ed.) 53 10557149
2005 Three-dimensional structure of Plasmodium falciparum Ca2+ -ATPase(PfATP6) and docking of artemisinin derivatives to PfATP6. Bioorganic & medicinal chemistry letters 52 15908211
2008 Synthesis, antimalarial activity, and intracellular targets of MEFAS, a new hybrid compound derived from mefloquine and artesunate. Antimicrobial agents and chemotherapy 51 18710907
1996 Analysis of a cation-transporting ATPase of Plasmodium falciparum. Molecular and biochemical parasitology 51 8813672
2013 Cytoplasmic free Ca2+ is essential for multiple steps in malaria parasite egress from infected erythrocytes. Malaria journal 50 23363708
2010 Purified E255L mutant SERCA1a and purified PfATP6 are sensitive to SERCA-type inhibitors but insensitive to artemisinins. The Journal of biological chemistry 49 20530490
2007 In vitro monitoring of Plasmodium falciparum drug resistance in French Guiana: a synopsis of continuous assessment from 1994 to 2005. Antimicrobial agents and chemotherapy 49 17954693
2010 Investigations into the role of the Plasmodium falciparum SERCA (PfATP6) L263E mutation in artemisinin action and resistance. Antimicrobial agents and chemotherapy 48 20566762
2008 Diversity of the sarco/endoplasmic reticulum Ca(2+)-ATPase orthologue of Plasmodium falciparum (PfATP6). Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 48 18359278
1996 Modulation of host cell intracellular Ca2+. Parasitology today (Personal ed.) 48 15275223
2012 Synthetic peroxides as potent antimalarials. News and views. Current topics in medicinal chemistry 45 22242847
2016 Evidence of a Mild Mutator Phenotype in Cambodian Plasmodium falciparum Malaria Parasites. PloS one 44 27100094
2017 Plasmid-free CRISPR/Cas9 genome editing in Plasmodium falciparum confirms mutations conferring resistance to the dihydroisoquinolone clinical candidate SJ733. PloS one 43 28542423
2022 A G358S mutation in the Plasmodium falciparum Na+ pump PfATP4 confers clinically-relevant resistance to cipargamin. Nature communications 42 36180431
2025 Optimization and Characterization of N-Acetamide Indoles as Antimalarials That Target PfATP4. Journal of medicinal chemistry 6 40228810
2025 Mutational analysis of an antimalarial drug target, PfATP4. Proceedings of the National Academy of Sciences of the United States of America 3 39773028
2025 Identification of α-Azacyclic Acetamide-Based Inhibitors of P. falciparum Na+ Pump (PfATP4) with Fast-Killing Asexual Blood-Stage Antimalarial Activity by Phenotypic Screening. ACS infectious diseases 2 40921413
2025 Validation of solvent proteome profiling for antimalarial drug target deconvolution. International journal for parasitology. Drugs and drug resistance 2 41308470
2025 Endogenous structure of antimalarial target Pf ATP4 reveals new class of apicomplexan P-type ATPase modulators. bioRxiv : the preprint server for biology 1 40060536
2025 A Soluble Expression Construct of the Isolated Catalytic Domain of Plasmodium falciparum ATP4 Exhibits ATPase Activity Independent of a γ-Phosphate Receiving Aspartate. Molecular microbiology 1 40091859
2025 Endogenous structure of antimalarial target PfATP4 reveals an apicomplexan-specific P-type ATPase modulator. Nature communications 1 41115914
2026 Identification of Compound 15 (MMV1581361) as a PfATP4 Inhibitor with Transmission-Blocking Activity and In Vivo Efficacy in a SCID Mouse Model of Malaria. Journal of medicinal chemistry 0 41670300
2026 Mechanisms of PfDNMT2 inhibition and PfATP6-mediated resistance to the antimalarial candidate SC83288 in Plasmodium falciparum. Nature communications 0 41786780
2026 High-throughput phenotypic screening identifies novel antimalarial scaffolds and target-associated chemotypes. International journal for parasitology. Drugs and drug resistance 0 42456602
2025 Identification of α-azacyclic acetamide-based inhibitors of P. falciparum Na + pump ( Pf ATP4) with fast-killing asexual blood-stage antimalarial activity by phenotypic screening. bioRxiv : the preprint server for biology 0 40475615
2025 Revisiting Plasmodium falciparum P-type ATPase 4 in malarial: ADMET, mutation effect, and molecular simulation studies of potential inhibitors. Journal of biomolecular structure & dynamics 0 40512986