DOI: 10.5061/dryad.2z34tmprm
title: pH-dependence of the Plasmodium falciparum chloroquine resistance transporter is linked to the transport cycle

description: 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.
description: 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
 <em>X. laevis</em> 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
 Ca<sup>2+</sup>-free ND96 buffer (96 mM NaCl, 2 mM KCl, 1 mM
 MgCl<sub>2</sub> 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 Na<sub>2</sub>HPO<sub>4</sub> 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
 CaCl<sub>2</sub>, 1 mM MgCl<sub>2</sub> buffered
 with 5 mM HEPES/NaOH, pH 7.5) supplemented with 100 mg
 l<sup>-1</sup> 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<sup>-1</sup>. 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<sup>-1 </sup>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
 <em>X. laevis</em> 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 <em>Xenopus</em> <em>laevis</em> oocyte
 membranes (8), using CharmmGUI (9) and Gromacs. For the docking of CQ to
 PfCRT Dd2, protein structures were<br>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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