pH-dependence of the Plasmodium falciparum chloroquine resistance transporter is linked to the transport cycle
Data files
Jun 16, 2023 version files 23.08 MB
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PfCRT_pH_raw_data.xlsx
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PfCRT_pH_Supplementary_information.pdf
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README.md
Abstract
This file contains the original data underpinning the following study:
The chloroquine resistance transporter, PfCRT, of the human malaria parasite Plasmodium falciparum displays a strong pH-sensitivity in the weakly acidic range. Consequently, PfCRT operates only at 60% of its maximal drug transport activity at the pH of 5.2 of the digestive vacuole, a proteolytic organelle from which PfCRT expels drugs that interfere with heme detoxification. Despite structural information, the molecular mechanism by which PfCRT senses pH changes has remained unclear. Here we show, by alanine-scanning mutagenesis and functional transport studies, that E207 plays a critical role in pH sensing. The E207A mutant displayed a pH-insensitive transport activity, while preserving drug substrate specificity. Replacement of E207 by Asp or His, but not by any other proteinogenic amino acid, reconstituted pH sensitivity. Molecular dynamics simulations and kinetics analyses suggest an allosteric binding model in which PfCRT can simultaneously accept both protons and chloroquine in a partial non-competitive manner, with increasing proton concentrations reducing the drug transport activity. Molecular dynamics simulations of the open-to-vacuole, the occluded and the open-to-cytosol conformation of PfCRT revealed a drastic relocation of E207 from a peripheral to an engaged location during the transport cycle, resulting in E207 forming a salt bridge with residue K80. We propose that the ionized carboxyl group of E207 acts as a hydrogen acceptor for interactions accelerating progression through the transport cycle and that the pH sensing is a by-product of this function.
Methods
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 Supplementary Table 7. The resulting DNA fragment was cloned into the SP64T 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 carefully 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). The obtained RNA was 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 4.5–7.0) supplemented with 10–500 µM unlabeled drug and [3H]chloroquine (50 nM), [3H]quinine (62.5 nM), [3H]quinidine (62.5 nM), or [3H]piperaquine (40 nM). The direction of radio-labeled drug transport is here from the mostly acidic extracellular solution (pH 4.5–7.0, as indicated) 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. 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. For pH 5.5, 6.0 and 6.5, ND96 was buffered with 5 mM MES/Tris base. For pH 4.5, 5 mM Homo-Pipes was used as a buffer and 5 mM HEPES for uptake at pH 7.0. 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 mM EDTA, 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 12% 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).
To model each conformation of PfCRT Dd2 (open-to-vacuole, occluded, open-to-cytoplasm), a total of 9 independent MD simulations of 200 ns each were performed: 3 with residue E207 protonated, 3 with residue E207 deprotonated, and 3 with residue E207 mutated to His. The protonation state of residues at pH 6 was determined using Propka 3.5 (7). PfCRT was embedded in a lipid bilayer consisting of cholesterol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) and 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) in the ratio 3:4:3 to represent Xenopus laevis oocyte membranes (8), using CharmmGUI (9) and Gromacs. For the docking of CQ to PfCRT Dd2, protein structures were
collected with a frequency of 10 ps from the last 150 ns of each of the three replicas of each conformation and each protonation state of E207, resulting in 45 protein structures for each conformation. The protein structures were clustered using the method gromos (10). Using AutoDock Vina (11), CQ was docked to the centers of the clusters. An exhaustiveness level of 8 and a cubic grid with a spacing of 0.375 Å, 80 points along each edge and center around the alpha carbon of E207 were used for docking. Bond rotations were allowed in the ligand, while the protein structures were kept rigid. For each cluster center, 20 ligand poses were generated by docking, and the top scoring pose (irrespective of the binding mode) was selected.
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