These data were generated to investigate the effect of point mutations in PfCRT on the kinetics of chloroquine and piperaquine binding and transport. The protein confers resistance to a range of quinoline and quinoline-like antimalarials after the acquisition of certain amino acid substitutions, which occurs as a result of improper antimalarial use. The change in prescription from chloroquine (CQ) to piperaquine (PPQ) in Southeast Asian countries led to the emergence of a ninth mutation over the PfCRT Dd2 isoform, which renders the parasites resistant to piperaquine but re-sensitizes them to chloroquine. Despite structural information, how these individual mutations influence such opposing changes in the parasite's susceptibility to the aforementioned drugs remains unknown. Here, we show by biochemical studies that any of the piperaquine resistance-conferring mutations H97Y, F145I, M343L or G353V, either reduce the affinity of PfCRT Dd2 for CQ (Km) or reduce the efficiency of the transport cycle (Vmax). In parallel, they increase the Vmax, reduce the Km, or do both, in the case of PPQ transport. We also probed the binding cavity of PfCRT Dd2 and that of PfCRT Dd2_F145I, and found that it can readily bind both CQ and PPQ simultaneously, in a partial noncompetitive mechanism. We confirmed the latter finding through molecular docking and molecular dynamics simulations, describing for the first time the binding sites for both drugs in the cavity of PfCRT Dd2. With this, we found that the pocket where CQ binds seems to require an aromatic side chain, which is normally provided by F145, or by Y97 in the PPQ resistance-conferring H97Y mutant. Lastly, we generated an unnatural double mutant carrying both the H97Y and the F145I mutations. The engineered transporter displayed non-Michaelis-Menten kinetics both for CQ and PPQ transport and instead revealed sigmoidal kinetics, typical of proteins that bind substrate cooperatively. We thus provide new insights into the organization of the substrate-binding cavity of PfCRT and into the evolution of PfCRT.

Site-directed mutagenesis of PfCRT-Dd2 was performed as described using the megaprimer method with overlap extension (1,2). The primer pairs used are listed in S3 Table. The resulting DNA fragment was cloned into the pSP64T expression vector and verified by sequencing (3). Oocytes were obtained as described previously (4). Briefly, adult female X. laevis frogs (NASCO) were anesthetized in a cooled solution of ethyl 3-amino benzoate methanesulfonate (0.1%, w/v), followed by a surgical removal of parts of the ovary. The ovary lobes were carefully dissected and small pieces of oocytes were incubated in Ca²⁺-free ND96 buffer (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂ buffered with 5 mM HEPES/NaOH, pH 7.5) supplemented with collagenase D (0.1%, w/v; Roche), BSA (0.5%, w/v), and 9 mm Na₂HPO₄ at 18 °C for 14–16 h while gently shaking.

After incubation, the oocytes were washed several times with ND96 buffer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂ buffered with 5 mM HEPES/NaOH, pH 7.5) supplemented with 100 mg l^-1 gentamycin). Defolliculated and healthy-looking stage V-VI oocytes were manually selected for RNA microinjection. The RNA was generated as follows: The codon-optimized sequences of PfCRT variants were cloned into the pSP64T expression vector. After vector linearization using the restriction endonucleases BamHI or SalI (New England Biolabs), transcription was performed using the in vitro SP6 mMessage mMachine Kit (Ambion) 32. The obtained RNA was kept at -80 °C and diluted with nuclease-free water to a concentration of 0.6 µg µl^-1. Precision-bore glass capillary tubes (3.5-inch glass capillaries; Drummond Scientific Co.) were pulled on a vertical puller (P-87 Flaming/Brown micropipette puller, Sutter Instrument Co.), graduated, and placed on a microinjector (Nanoject II Auto-Nanoliter Injector, Drummond Scientific Co.). 50 nl of nuclease-free water alone (control oocytes) or 50 nl of 0.6 µg µl^-1 RNA were injected under stereomicroscopic control. Injected oocytes were incubated for 46–72 h at 18°C with twice-daily buffer changes before usage in experiments.

Drug uptake assays were performed as described previously (1,3,4,5). Briefly, oocytes were incubated for 1 h at room temperature in ND96 buffer (pH 6.0) supplemented with 10–500 µM unlabeled drug and [3H]chloroquine (50 nM) or [3H]piperaquine (40 nM). Where indicated, a second, unlabeled drug (chloroquine or piperaquine) was also present in the buffer. The direction of radio-labeled drug transport is from the mostly acidic extracellular solution (pH 6.0) into the oocyte cytosol (pH ~ 7.2), which corresponds to the efflux of drug from the acidic digestive vacuole (pH 5.2) into the parasite cytoplasm (pH 7.2). For determination of the amount of drug uptake for each oocyte, the reaction medium was removed and the oocytes were washed three times with 2 ml of ice-cold ND96 buffer. Each oocyte was individually transferred to a scintillation vial containing 5% sodium dodecyl sulfate (200 µl) for lysis. The radioactivity of each sample was measured using a liquid scintillation analyzer Tri-Carb 4910 TR (PerkinElmer). Oocytes that were injected with water instead of RNA were analyzed in parallel, representing the uptake of a drug by diffusion and/or endogenous permeation pathways. The resulting value represents the “background“ level of drug accumulation in oocytes (in-depth discussion in 22). The specific PfCRT-mediated uptake was determined by subtracting the corresponding background value measured in water-injected oocytes from that of PfCRT-expressing oocytes. In all cases, at least three separate experiments were performed on oocytes from different frogs (biological replicate), and for each condition in an experiment, measurements were made from 10 oocytes per treatment.

Western blot analysis of oocytes was performed as described previously (6). Briefly, 3 days after RNA injection, total lysates were prepared from X. laevis oocytes by adding 20 µl of radioimmunoprecipitation assay-lysis buffer (10 mM HEPES-Na, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (CompleteTM, Roche Applied Science). After removing of cellular debris by centrifugation, lysates were mixed with 2x sample buffer (250 mM Tris, pH 6.8, 3% SDS, 20% glycerol, 0.1% bromphenol blue). Then, the extracts were size-fractionated using 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The following antibodies were used: guinea pig anti-PfCRT antiserum (raised against the N terminus of PfCRT (MKFASKKNNQKNSSK); 1:1000 dilution; Eurogentec), donkey anti-guinea pig POD (1:10,000 dilution; Jackson ImmunoResearch Laboratories), monoclonal mouse anti-a-tubulin (1:1000 dilution; clone B-5-1-2) and goat anti-mouse POD (1:10,000 dilution; Jackson ImmunoResearch Laboratories). All antibodies were diluted in 1% (w/v) BSA in PBS. The signal was captured with a blot scanner (C-DiGit) and quantified using Image Studio Digits version 4.0 (Licor).

Immunofluorescence of oocytes was done as previously described (7). Briefly, 3 days after RNA injection, oocytes were fixed with 4% (v/v) paraformaldehyde in PBS for 4h at room temperature. After three washing steps with 3% (w/v) BSA in PBS, fixed oocytes were incubated with guinea pig anti-PfCRT antiserum (1:500 dilution) overnight at 4°C. Three subsequent washing steps with 3% (w/v) BSA in PBS were followed by incubation with wheat germ agglutinin Alexa Fluor 633 (5 µg ml^-1) for 10 min at 4°C. After three extra washing steps as before, oocytes were analysed by fluorescence microscopy using a Zeiss LSM 510 confocal microscope, and images were processed with Fiji.

For the docking of chloroquine and piperaquine to PfCRT, the structure of PfCRT Dd2 was modeled based on homology from the deposited structure of PfCRT 7G8 using the SWISS-MODEL Protein Modelling server (8). The protein was prepared for docking at pH 6 using the Protein Preparation Wizard in Maestro (9). The ligands CQ and PPQ were prepared and ionized at pH 6.0 with LigPrep (10). Induced-fit docking (11) was performed using Q235, S90, Q156,L221, W352, L83, and F145 as centroids, generating 10 poses for each ligand. The first docking pose of PPQ and the third pose of CQ were merged and embedded into a POPC (1-palmitoyl-2-oleoyl-phosphatidylcholine) lipid bilayer (12,13). The systems were solvated in a periodic box of TIP3P water molecules and neutralized at an ion concentration of 150mM NaCl. Molecular dynamics simulations were run using the Amber20 software (14). The systems were minimized with 10 consecutive runs (1000 steps each) of decreasing restraints from 1000 to 0.01kcal mol^-1 A-2 to the heavy atoms and an additional 1000 steps without restraints. Heating was performed in two stages of 200ps, first to 100K using restraints of 100kcal mol^-1 A-2 with the Langevin thermostat, and then up to 310K with restraints of 5kcal mol^-1 A-2. A 4ns equilibration with restraints of 5kcal mol^-1 A-2 was then performed, and 10 consecutive simulations (5ns each) without restraints were performed to equilibrate the system's periodic boundary dimensions. A production of 1µs under the NPT ensemble was run. A time step of 2fs was used for all simulations and bonds with hydrogens were constrained using the SHAKE algorithm. 

1 Summers, R. L. et al. Diverse mutational pathways converge on saturable chloroquine transport via the malaria parasite's chloroquine resistance transporter. Proc Natl Acad Sci U S A 111, E1759-1767 (2014).

2. Zheng, L., Baumann, U. & Reymond, J. L. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res 32, e115 (2004).

3. Martin, R. E. et al. Chloroquine transport via the malaria parasite's chloroquine resistance transporter. Science 325, 1680-1682 (2009).

4. Bakouh, N. et al. Iron is a substrate of the Plasmodium falciparum chloroquine resistance transporter PfCRT in Xenopus oocytes. J Biol Chem 292, 16109-16121 (2017).

5. Bellanca, S. et al. Multiple drugs compete for transport via the Plasmodium falciparum chloroquine resistance transporter at distinct but interdependent sites. J Biol Chem 289, 36336-36351 (2014).

6. Rotmann, A. et al. PfCHA is a mitochondrial divalent cation/H+ antiporter in Plasmodium falciparum. Mol Microbiol 76, 1591-1606 (2010).

7. Sanchez, C.P. et al. Phosphomimetic substitution at Ser-33 of the chloroquine resistance transporter PfCRT  reconstitutes drug responses in Plasmodium falciparum. J Biol Chem. 294(34):12766-12778 (2019).

8. Waterhouse, A. et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids  Res. 46(W1):W296-w303 (2018).

9. Sastry, G.M. et al. Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J Comput Aided Mol Des. 27(3):221-234 (2013).

10. Shelley, J.C. et al. Epik: a software program for pK( a ) prediction and protonation state generation for drug-like molecules. J Comput Aided Mol Des. 21(12):681-691 (2007).

11. Sherman, W. et al. Use of an induced fit receptor structure in virtual screening. Chem Biol Drug Des. 67(1):83- 84 (2006).

12. Jo, S. et al. CHARMM-GUI: a web-based graphical user interface for CHARMM. J Comput Chem. 29(11):1859-1865 (2008)

13. Dickson, C.J. et al. Lipid14: The amber lipid force field. J Chem Theory Comput. 10(2):865-879 (2014).

14. Case, D.A. et al. AMBER: University of California, San Francisco, CA (2014).