DOI: 10.5061/dryad.prr4xgxr5
title: PfCRT mutations conferring piperaquine resistance in falciparum malaria shape the kinetics of quinoline drug binding and transport

description: 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.
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 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 <em>X</em>.
 <em>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 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) 32. The obtained RNA was kept at -80 °C and 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 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 <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% 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<sup>-1</sup>) 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<sup>-1</sup>
 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<sup>-1</sup> A-2 with the Langevin
 thermostat, and then up to 310K with restraints of 5kcal
 mol<sup>-1</sup> A-2. A 4ns equilibration with restraints of
 5kcal mol<sup>-1</sup> 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
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