On the Mechanism of Chloroquine Resistance in
Plasmodium falciparum
Mauro Chinappi1., Allegra Via1., Paolo Marcatili1, Anna Tramontano1,2*
1 Department of Biochemical Sciences, Sapienza University, Rome, Italy, 2 Istituto Pasteur, Fondazione Cenci Bolognetti, Sapienza University, Rome, Italy
Abstract
Resistance to chloroquine of malaria strains is known to be associated with a parasite protein named PfCRT, the mutated
form of which is able to reduce chloroquine accumulation in the digestive vacuole of the pathogen. Whether the protein
mediates extrusion of the drug acting as a channel or as a carrier and which is the protonation state of its chloroquine
substrate is the subject of a scientific debate. We present here an analytical approach that explores which combination of
hypotheses on the mechanism of transport and the protonation state of chloroquine are consistent with available
equilibrium experimental data. We show that the available experimental data are not, by themselves, sufficient to conclude
whether the protein acts as a channel or as a transporter, which explains the origin of their different interpretation by
different authors. Interestingly, though, each of the two models is only consistent with a subset of hypotheses on the
protonation state of the transported molecule. The combination of these results with a sequence and structure analysis of
PfCRT, which strongly suggests that the molecule is a carrier, indicates that the transported species is either or both the
mono and di-protonated forms of chloroquine. We believe that our results, besides shedding light on the mechanism of
chloroquine resistance in P. falciparum, have implications for the development of novel therapies against resistant malaria
strains and demonstrate the usefulness of an approach combining systems biology strategies with structural bioinformatics
and experimental data.
Citation: Chinappi M, Via A, Marcatili P, Tramontano A (2010) On the Mechanism of Chloroquine Resistance in Plasmodium falciparum. PLoS ONE 5(11): e14064.
doi:10.1371/journal.pone.0014064
Editor: Vladimir Brusic, Dana-Farber Cancer Institute, United States of America
Received June 16, 2010; Accepted October 28, 2010; Published November 19, 2010
Copyright: ß 2010 Chinappi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by King Abdullah University of Science and Technology (KAUST) - Award No. KUK-I1-012-43 (http://www.kaust.edu.sa/),
Human Frontier Science Program (HFSP) - RGP0054/2006-C grant (http://www.hfsp.org/), and Fondazione Roma (http://www.fondazioneroma.it/). The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Anna.Tramontano@uniroma1.it
. These authors contributed equally to this work.
proteolysis [5,6], preventing its incorporation into the haemozoin
crystal [2,7,8,9,10]. The free haematin seems to interfere with the
parasite detoxification processes and thereby damage the plasmodium membranes [11].
Chloroquine sensitive parasites (CQS) accumulate much more
chloroquine in the DV than chloroquine resistant strains (CQR)
[4,12,13]. Recent studies have associated the reduced chloroquine
accumulation observed in the parasite vacuole of resistant strains
[12] with point mutations in the gene encoding for the P. falciparum
chloroquine resistance transporter (PfCRT) protein (for a review
see [14,15]). PfCRT is localized in the digestive vacuole
membrane and contains 10 predicted membrane-spanning
domains [16,17]. CQR phenotype isolates have all been found
to carry the PfCRT critical charge-loss mutation K76T or, in two
single cases, K76N or K76I [18,19,20,21]. Another mutation,
S163R, restores the chloroquine sensitivity of CQR parasites
[22,23]. The K76T amino acid mutation might allow the
interaction of PfCRT with the positively charged chloroquine
(CQ+ or CQ++) and allow its exit from the vacuole, with the net
result of decreasing the chloroquine concentration within the DV
[16,24]. The single amino acid change S163R, by reintroducing a
positive charge, is thought to block the leak of charged chloroquine
from the DV, thus restoring chloroquine sensitivity [22,23]. In a
recent work, Martin and collaborators [25] were able to express
both wild-type and resistant forms of PfCRT on the surface of
Introduction
In the last decades, due to its effectiveness and reasonable cost,
chloroquine has represented the best and more widely used
antimalarial drug. Unfortunately, within a decade of its introduction, P. falciparum parasite resistance to chloroquine was observed
in most of the malaria-endemic countries. Nowadays, insurgence
of resistance against chloroquine is a considerable hurdle for
malaria control [1].
In its erythrocyte stage, P. falciparum invades the red blood cells
where it forms a lysosomal isolated acidic compartment known as
the digestive vacuole (DV). In the erythrocyte, the parasite grows
by ingesting haemoglobin from the host cell cytosol and depositing
it in the DV, where the protein is degraded to its component
peptides and heme, which is incorporated into the inert and
harmless crystalline polymer hemozoin [2].
Chloroquine is a diprotic weak base and, at physiological pH
(,7.4), can be found in its un-protonated (CQ), mono-protonated
(CQ+) and di-protonated (CQ++) forms. The uncharged chloroquine is the only membrane permeable form of the molecule and it
freely diffuses into the erythrocyte up to the DV. In this
compartment, chloroquine molecules become protonated and,
since membranes are not permeable to charged species, the drug
accumulates into the acidic digestive vacuole [3,4] where it is
believed to bind haematin, a toxic byproduct of the haemoglobin
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Chloroquine Resistance
[31]. On the other hand, Bray and colleagues [29] propose that
the trans-stimulation data reported by Sanchez et al cannot by
themselves be used to conclude whether the chloroquine transport
is in the inward or outward direction: stimulation of [3H]-CQ
uptake could indeed be due to acceleration of the transporter
cycle by the outgoing unlabelled chloroquine or, as Sanchez et al
assert, it could result from reduced efflux of [3H]-CQ due to the
carrier competitive inhibition from the pre-loaded unlabelled
CQ. In the latter model, labeled and unlabelled chloroquine
should be on the same side of the membrane when they interact
with the carrier, i.e. they are mixed together. In order to verify this
hypothesis, Bray et al [29] incubated CQR lines with premixed
chloroquine (labeled and unlabelled), but did not observe transstimulation of chloroquine uptake, thus suggesting that labeled
and unlabelled chloroquine must be on opposite sides of the
membrane for the trans-stimulation effect to take place, i.e.
transport of unlabelled chloroquine via the carrier would be in the
outward direction while labeled chloroquine transport would
occur in the inward direction; in other words, mutant PfCRT
would act as a bidirectional carrier, which is not compatible with
an active efflux pump. In particular, these authors conjecture that
trans-stimulation results might also be explained in terms of a
gated channel.
Many authors measured chloroquine efflux from CQS and
CQR isolates [27,29,32,33] under different conditions: in presence
and absence of glucose, with or without proton gradient
uncoupling, with or without Verapamil, an L-type calcium
channel blocker of the Phenylalkylamine class. The results of
these experiments have been interpreted in different ways by
different authors and did not lead to a consensus view about the
nature of PfCRT.
Sanchez et al [34] studied the kinetics of chloroquine efflux in
‘reverse varying-trans’ conditions [31] from CQR and CQS
isolates. This procedure investigates whether extracellular unlabelled chloroquine would stimulate the release of pre-loaded [3H]CQ. These authors expected that, in the presence of an active
carrier, trans chloroquine should increase the initial efflux rate.
They found an increasing initial efflux rate for both CQR and
CQS lines and accordingly proposed that both CQR and CQS
parasites possess a carrier of chloroquine with different transport
properties.
It should appear clear from this survey that qualitative
interpretations of the experimental findings are insufficient to
draw conclusions about the nature of PfCRT and that more
quantitative analyses are required.
A quantitative model cannot be derived from transient
experiments because kinetic parameters, such as the rate of the
vacuole pH equilibration during chloroquine uptake and the
kinetic constants of chloroquine-hemozoine binding, are unknown
and impossible to extract from the available data in an
unambiguous way. The only data that can be used to derive a
quantitative model without making an unreasonable number of
hypotheses on the unknown parameters are those measured at
equilibrium.
As we will show, the analytical model that we developed and
used here indicates that equilibrium data are compatible with both
the carrier and channel model for PfCRT, which explains why
they could be interpreted differently by different authors. On the
other hand, the carrier and channel hypotheses are only
compatible with specific assumptions on the protonation state of
the transported species and of the species binding to haeme of
haeme-related molecules in the vacuole. For example, a carrier
model is only compatible with the data if the transported molecule
is protonated.
Xenopus laevis oocytes and clearly demonstrated that chloroquine
resistance is due to the direct transport of a protonated form of the
drug out of the parasite vacuole via the K76T PfCRT mutant.
Interestingly, they also showed that the introduction of the K76T
single mutation in PfCRT of CQS parasites is necessary but not
sufficient for the transport of chloroquine via PfCRT. These
evidences are however compatible with two alternative models for
PfCRT [26]: (1) the channel model (i.e. a passive channel that
enables charged chloroquine to leak out of the food vacuole down
its electrochemical gradient) or (2) the carrier model (i.e. an active
efflux carrier extruding chloroquine from the food vacuole).
Several experimental set-ups have been used to answer the
question of whether PfCRT is a channel or a carrier, namely
measures of chloroquine accumulation, trans-stimulation and
measures of chloroquine efflux. However the available data have
been interpreted in different ways by different authors and the
debate about the nature of PfCRT is still ongoing.
Sanchez and colleagues showed that chloroquine accumulation
is energy dependent in both CQR and CQS [27]. These authors
monitored the time course of labeled chloroquine uptake in the
absence and in the presence of glucose. Glucose was added 20 min
after choloroquine addition (i.e. when the stationary state was
reached). They found that, after glucose addition, the time courses
of choroquine uptake were markedly different in CQS and CQR:
chloroquine accumulated to an increased extent in the CQS
strain, but decreased in the CQR strain. A similar experiment was
repeated by the same authors in 2004 [28] using a broader range
of different antimalarial drugs. The authors concluded that the
data are compatible with most models that attempt to account for
chloroquine resistance and that some energy-dependent mechanism leads to loss of chloroquine from CQR cells and to its
accumulation in CQS cells.
Bray et al [29], in 2006, measured the Cellular Accumulation
Ratio (CAR) of chloroquine in six experimental conditions,
namely in sensitive and resistant strains, in the absence and
presence of carbonylcyanide p-trifluoromethoxyphenylhydrazone
(FCCP), a ionophoric uncoupling agent, and in the absence and
presence of glucose. In particular they found that, in absence of
glucose, chloroquine the CAR is equal in CQS and CQR strains
(,700), reaching a level that is approximately intermediate
between that observed in CQS (,1200) and CQR (,350) strains
in the presence of glucose. They used several different Plasmodium
strains and showed that, in the absence of FCCP, i.e. when the pH
of the vacuole is lower than the external pH, the chloroquine CAR
is three to four times higher (about 1200 versus about 350) in
sensitive strains with respect to resistant strains, while addition of
FCCP abolishes the differences leading to a CAR value of about
700 in both cases. They also demonstrate that, in the absence of
glucose, the CAR is identical to that obtained in the presence of
FCCP suggesting that the energy provided by the glucose is
needed to maintain the pH difference between the cytoplasm and
the DV. According to the authors, the hypothesis that PfCRT is an
active efflux carrier does not appear to fully explain their findings.
In this hypothesis, in fact, a single mutation would transform an
energy-dependent chloroquine uptake process in an energydependent chloroquine efflux process. Therefore they favor the
hypothesis that the chloroquine movement through PfCRT is not
an active process.
Trans-stimulation of labeled chloroquine ([3H]-CQ)) uptake
after the parasites were pre-loaded with increasing concentrations
of unlabelled chloroquine [27,28,29,30] was observed in CQR
strains and not in CQS isolates. Sanchez and collaborators
conclude that the trans-stimulation phenomenon is unequivocally
characteristic of saturable, carrier-mediated transport systems
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Chloroquine Resistance
Another route to understand the nature of the macromolecule is
to study its evolutionary relationship with other proteins of known
function. Also in this case, data interpretation is controversial.
Previous computational analyses of PfCRT [14,16] suggested that
PfCRT belongs to the drug/metabolite trasporter (DMT)
superfamily, whereas other studies proposed that it resembles to
ClC chloride channels [35].
Here, we use state-of-the-art bioinformatics tools to identify
PfCRT homology relationships and provide evidence that it is
indeed a member of the DMT superfamily This finding is also
supported by the observation that a three-dimensional model of
the protein based on a DMT-like fold is consistent with
experimental data about the mutations involved in insurgence
and reversion of CQ resistance in Plasmodium.
By combining this latter conclusion with the results of the
analytical method, we propose that PfCRT is a carrier of CQ+,
CQ++ or both and that either all chloroquine species or only the
uncharged one can bind hame of hame related species inside the
vacuole.
Table 1. Values of the chloroquine cellular accumulation ratio
in sensitive and resistant strains, in the presence and absence
of FCCP, as estimated from figures 1A and 2A in Bray et al [29].
2FCCP
+FCCP
Sensitive strain (CQS) GC03 strain
,1200 (B)
,700 (A)
Resistant strain (CQR) Dd2 strain
,350 (C)
,700 (D)
doi:10.1371/journal.pone.0014064.t001
[CQ:HM]DV = a [CQTOT]DV, where [CQTOT] = [CQ]+[CQ+]+
[CQ++]. The latter representing the case of chloroquine binding
HM regardless of its protonation state.
H2) The concentration of the complex chloroquine:HM
increases non linearly with the concentration of the binding
chloroquine form in the vacuole and reaches saturation at
concentrations of chloroquine above a given threshold. If
we define the threshold concentrations as tHM,CQ, tHM,CQ+, tHM,CQ++
or tHM,CQtot or simply tHM when the species is clear from the context,
the above hypothesis can be expressed as: [CQ:HM]DV = f([CQ]DV)
that, for CQDV.tHM,CQ, reads [CQ:HM]DV = constant. Similar
expressions hold for [CQ:HM]DV = f([CQ+]DV), [CQ:HM]DV =
f([CQ++]DV) and [CQ:HM]DV = f([CQTOT]DV).
As far as PfCRT is concerned, the two possible cases are:
Results
We selected to use the data from the Cellular Accumulation
Ratio (CAR) experiments described by [29] because, as mentioned
above, the experimental conditions (stable and controlled pH
values) allow the model to be built using a number of parameters
comparable with the number of observations.
Our approach consists in testing the consistency of all plausible
hypotheses about the binding mode of the drug to the heme
related species inside the vacuole and the mechanism of action of
PfCRT with experimental data. Our reasoning does not require
any assumption on which heme form or heme related molecule
binds to chloroquine and, for this reason, we refer to the heme
related species bound by chloroquine inside the vacuole as HM.
Two mechanisms are thought to be involved in chloroquine
accumulation into the P. falciparum vacuole: acidic trapping due to
low vacuolar pH and chloroquine binding to heme or heme
related species. It is reasonable to assume that PfCRT does not
directly affect the molecular mechanism of chloroquine-HM
binding. Recent studies [36,37,38] indicate that the vacuolar pH
of CQS and CQR strains are similar, hence the reduction of
chloroquine accumulation in resistant strains cannot be explained
in terms of different acidic trapping and PfCRT must be directly
involved in releasing chloroquine out of the vacuole. A proof of
this hypothesis has been recently provided by Martin and
collaborators [25].
J1) PfCRT acts as a passive channel for CQ+ (or CQ++, or
both). and thereby the outward flux of chloroquine across the
vacuole membrane due to PfCRT, JPfCRT, only depends upon the
difference in concentration on the two sides of the vacuolar
membrane and on the membrane potential. For instance, if the
membrane potential is zero and the channel allows CQ+ to move
out of the DV, JPfCRT = f([CQ+]DV2[CQ+]e), the suffix ‘‘e’’
indicating the plasmodium cytoplasm. As shown in the Text S1,
section S1, being the vacuole membrane freely permeable to unprotonated chloroquine CQ, the hypothesis that PfCRT acts as a
channel for CQ alone can be immediately discarded since it is not
consistent with the experimentally observed differences between
CQR and CQS.
J2) PfCRT is a carrier for CQ, (or CQ+, or CQ++). In this
case, it is reasonable to assume that the flux through the channel is
a linear function of the CQ (or CQ+, or CQ++) concentration for
concentration values below a given threshold and a constant
above the threshold concentration. Let us define the threshold
concentrations as tPfCRT,CQ, tPfCRT,CQ+, or tPfCRT,CQ++ or simply
tPfCRT when the species is clear from the context. The above
hypotheses can be expressed as:
The analytical approach
J2aÞ JPfCRT ~l ½CQDV ; for ½CQDV vtPfCRT,CQ
In this study we formulate an analytical model describing
different combinations of the two sets of hypotheses described
below, the first related to the mode of chloroquine binding to HM,
the second to the mechanism of action of the mutated PfCRT. We
test all possible combinations of these hypotheses for their
consistency with the available experimental data provided by
[29] and summarized in Table 1. Abbreviations used throughout
the manuscript are reported in Table 2.
Concerning the chloroquine binding to HM we consider two
possibilities:
J2bÞ JPfCRT ~constant ; for ½CQDV wtPfCRT,CQ
similar expressions hold for JPfCRT = f([CQ+]DV), JPfCRT =
f([CQ++])DV.
The procedure adopted to test all combinations of the above
hypotheses for their consistency with the available experimental
data makes use of the analytical expressions for the membrane
equilibrium, for the Cellular Accumulation Ratio (CAR) of
chloroquine, which is the quantity measured in the experiments
described in [29] and for the basis dissociation equilibrium.
H1) The concentration of the complex chloroquine:HM
inside the vacuole, [CQ:HM]DV, linearly increases with the
concentration of the binding form of chloroquine in the
vacuole. According to which chloroquine species reacts with
HM, this can be expressed as [CQ:HM]DV = a [CQ]DV or
[CQ:HM]DV = a [CQ+]DV, or [CQ:HM]DV = a [CQ++]DV or
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CAR
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Table 2. List of abbreviations used throughout the paper.
Abbreviation
Description
CQS
Cloroquine Sensitive Strain
CQR
Cloroquine Resistant Strain
CAR
Cellular Accumulation Ratio
CQ
un-protonated form of chloroquine
CQ+
mono-protonated form of chloroquine
CQ++
di-protonated form of chloroquine
CQTOT
CQ+CQ++CQ++
CQ*
it is used in equations that hold for all four forms of chloroquine (CQ, CQ+, CQ++
and CQTOT)
[3H]-CQ
labeled chloroquine
DV
Digestive Vacuole
HM
heme related species bound by chloroquine inside the vacuole
[CQ], [CQ+], [CQ++], [CQTOT]
Concentration of CQ, CQ+, CQ++, CQTOT
[CQ:HM]DV
concentration of the complex chloroquine:HM inside the vacuole
[H+]
concentration of H+
FCCP
carbonylcyanide
p-trifluoromethoxyphenylhydrazone, a ionophoric uncoupling agent
JPfCRT
chloroquine flux through PfCRT
HMM
Hidden Markov Model
doi:10.1371/journal.pone.0014064.t002
Finally, the relationships between the concentrations of the
three forms of chloroquine given by the two-base dissociation
equilibrium are:
The membrane equilibrium equation is:
Pcq (½CQe {½CQDV )~JPfCRT
ð1Þ
where Pcq is the membrane permeability to unprotonated
chloroquine; equation (1) has been obtained taking into account
that the system has reached a steady state in the analysed
experimental conditions. Consequently, the net chloroquine flux
across the membranes is zero. The only form of chloroquine for
which the erythrocyte and the external plasmodium membranes
are permeable is the un-protonated one and this implies that CQ
concentrations are the same on the two sides of these membranes.
The expression for CAR is:
CAR~
½Cin
½C VDV z½Ce (Vin {VDV )
~ DV
½Ce Vin
½Cout
½CQz ~
½CQzz ~
ð2Þ
ð3Þ
being [CQ:HM]DV the concentration of chloroquine bound to
HM.
½Ce ~½Cout ~ CQTOT e ~½CQe z½CQz e z½CQzz e ð4Þ
is the concentration of chloroquine outside the vacuole, [C]in is the
average chloroquine concentration in the infected erythrocyte, Vin
is the volume of the infected erythrocyte and VDV is the volume of
the digestive vacuole. The analytical derivation of eqs (1) and (2) is
detailed in Text S1, sections S2 and S3, respectively. The proof of
the first equality of eq (4) ([C]e = [C]out) is reported in Text S1,
section S4.
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ð5Þ
½CQ½H z 2 ½CQ10{2pH
~
k’’
k’’
ð6Þ
with 1/k9 = (1/k2+1/k2) and k0 = k1k2 and k1 and k2 are the two
dissociation constants.
Experimentally determined values for the constants used in Eqs
(2) to (6) are reported in Table 3.
Figure 1 summarizes the various hypotheses tested here. The
columns refer to the mode of binding of the chloroquine to HM,
assuming either that the experimental data have been obtained in
non saturation conditions (linear regime, hypothesis H1) or that
the concentration of the chloroquine species is above the
(unknown) saturation concentration for HM (saturation conditions, hypothesis H2). The rows are related to assumptions about
the mutated PfCRT function considering the possibility that the
latter is a channel (first row, hypothesis J1) or an active carrier
(remaining rows, hypotheses J2) and, in the case of active carrier,
considering the possibility that the considered chloroquine species
concentration into the vacuole is or is not above the (unknown)
threshold needed to saturate PfCRT.
In the following we will show the procedure used to test the
hypotheses summarized in Figure 1. We use the symbol CQ* in
equations that hold for all four forms of chloroquine (CQ, CQ+,
CQ++ and CQTOT).
We examined the consequences of each of the hypotheses
reported in Figure 1. As a general strategy, we used the values of
where the total concentration of chloroquine inside the vacuole is:
½CDV ~½CQDV z½CQz DV z½CQzz DV z½CQ : HMDV
½CQ½H z ½CQ10{pH
~
k’
k’
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Chloroquine Resistance
Table 3. Values of the parameters used throughout the
paper.
Parameter
Description
Value
Reference
Cout = Ce
external chloroquine concentration
2 nM
[29]
k1
chloroquine dissociation constants
1028.1
[53]
k2
chloroquine dissociation constants
10210.2
[53]
Vin
volume of the erythrocyte
80 fL
[10]
VDV
volume of the vacuole
4 fL
[10]
Pcq
vacuole membrane permeability
7.5 cm/s
[54]
[55]
pHe
external (physiological) pH
7.4
[29]
pHDV
vacuole pH
4.5–4.9
5.1860.05
5.4–5.5
[36]
[37]
[38]
ð8Þ
case 3 : ½CDV ~ CQTOT DV za½CQzz DV
ð9Þ
case 4 : ½CDV ~(1za) CQTOT DV
ð10Þ
corresponding to columns 5, 6, 7 and 8 of Figure 1, respectively.
Combining these equations with (2), (3), (4), (5), and (6) an
expression for CAR as a function of the binding constant a and of
pHDV can be obtained. For instance, in case 1 (eq. (7)), we have:
CAR~
doi:10.1371/journal.pone.0014064.t003
½CQDV
Ve zVDV
½CQTOT e
½H z DV ½H z 2DV
z
1zaz
k’
k’’
Ve zVDV
!
ð11Þ
where Ve = Vin2VDV.
Let us consider the behavior of CQS strains. In this case,
[CQ]DV = [CQ]e ((eq-S3) Text S1, section S2) and, in the presence
of FCCP, [H+]DV is equal to [H+]e. Hence, by substituting the
known value of CAR for sensitive strains (experiment A in
Table 1), we can compute the parameter a. Knowing a and using
the experimental value of CAR in the absence of FCCP
(experiment B in Table 1) we can solve the second order equation
(11) in pHDV, which is found to have only one positive solution.
The same procedure can be applied to equations (8–10) to derive
the equivalent of (11) for cases 2–4 (see also Text S1, section S5).
The results for the four cases are:
CAR in experiments A, B and C reported in Table 1 (CARA,
CARB and CARC, respectively) to calibrate the model, inferred a
CAR value for experiment D (CARD) and compared it with the
experimental one. In some cases, the value of CARA and CARB
was used to infer the value of the vacuolar pH (pHDV)
subsequently compared with the experimental one. When the
results show that a hypothesis is inconsistent with the experimental
data and has to be discarded, the corresponding cell in Figure 1 is
shaded. Inferred values of CARD and their agreement or
disagreement with experimental values are shown in Figure 2B.
Test of hypothesis H1 ([CQ:HM]DV = a [CQ*]DV). Here,
we use the values of CARA and CARB to infer the pHDV value.
Notice that, since CARA and CARB refer to CQS strains, here we
are not making any assumption on PfCRT.
Let us consider the hypothesis that the concentration of the
complex CQ*:HM linearly increases with the vacuolar concentration of CQ*, i.e. hypothesis H1 corresponding to the twenty
cells C1–5,5–8 in Figure 1 in the columns labeled as ‘‘Linear
regime’’. We can have four different expressions for the total
chloroquine accumulated in the vacuole [C]DV depending on
which chloroquine form reacts with HM, namely
case 1 : ½CDV ~ CQTOT DV za½CQDV
case 2 : ½CDV ~ CQTOT DV za½CQz DV
ð7Þ
Case 1 : a~5:311 107
pHDV ~5:36
Case 2 : a~8:352 104
pHDV ~7:17
Case 3 : a~1:680 104
pHDV ~7:28
Case 4 : a~1:398 104
pHDV ~7:27
Figure 1. Summary of the hypotheses analysed throughout the manuscript. The columns refer to the mode of binding of the chloroquine
to HM, the rows to mutated PfCRT, considering the possibility that the latter is a channel or an active carrier. Shaded cells correspond to combination
of hypotheses inconsistent with the analysed data.
doi:10.1371/journal.pone.0014064.g001
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Chloroquine Resistance
Figure 2. Graphical representation of the results of the analytical model in comparison with the experimental data. A) Computed
values for the pH of the vacuole in the hypothesis that the concentration of the complex of HM with the indicated CQ species linearly increases with
the concentration of the ligand (first four columns) or that the system is at saturation (last column). ALL refers to either CQ, CQ+, CQ++, CQTOT. Orange
shaded area indicates the range of experimental values and their uncertainty measured by different authors in different experiments. As described in
the text, the calculation only uses the CAR for sensitive strains (experiments A and B in Table 1) and therefore does not require any hypothesis on the
nature of the mutated PfCRT. B) Computed values of the CAR compared with observed values obtained in experiment D (see Table 1). The different
hypotheses are shown in the table at the bottom. The first row refers to the cells of the table in Figure 1. The second row reports the tested
hypotheses on the mechanism of PfCRT (channel or carrier), on whether the binding of PfCRT with the indicated species is in the linear regime (linear)
or at saturation (saturated) and on the protonation state of the transported molecule (CQ: neutral; CQ+: mono-protonated; CQ++: di-protonated). The
third row refers to the tested hypotheses for the binding of the listed chloroquine species (CQ, CQ+, CQ++ or CQTOT; ALL refers to either CQ, CQ+, CQ++,
CQTOT.) with HM in the hypotheses that the latter complex is at saturation or in the linear regime. Grey shaded cells indicate that the computed values
are incompatible with the experimental ones. The orange shaded area indicates the range of experimental values and their experimental error.
Dashed arrows indicate that the model provides an upper limit for the value. Question marks indicate that no conclusion can be derived using the
indicated combination of hypotheses.
doi:10.1371/journal.pone.0014064.g002
Only the pHDV value obtained in case 1 is compatible with the
experimental data for pHDV (Figure 2A and Table 3) and therefore
we can conclude that, if the data are obtained in non saturating
conditions for HM binding, the non protonated form of chloroquine
binds to HM, regardless of the mechanism of action of the mutated
PfCRT. The C1–5,6–8 cells in Figure 1 are therefore shaded since
they correspond to cases not compatible with the experimental data.
that pHDV = pHe, i.e. that the the concentrations of the three
chloroquine forms are the same inside and outside the vacuole.
Combining this observation with eq. (12) and the membrane
balance equation (eq. (1)), we obtain that [CQ]DV,D = [CQ]e,
[CQ+]DV,D = [CQ+]e and [CQ++]DV,D = [CQ++]e, i.e. both the
chloroquine diffusive flux through the vacuolar membrane and
through PfCRT (eq (12)) are zero (for detailed calculations see
Text S1, section S6). These findings imply that CARD = CARA,
which is consistent with the experimental data (Figure 2B).
Accordingly, cell C1,5 of Figure 1 and Figure 2B is not shaded.
Notice that, as a by-product of our calculations, the JPfCRT
permeability can be explicitly calculated in the hypothesis that
f(x) is a linear function of ([CQ+]DV2[CQ+]e) (see Text S1,
section S7).
The argument used to demonstrate that CARD = CARA when
the membrane potential is zero also holds if the membrane
potential is different from zero. According to [26,29] we assume
that the DV membrane potential is mainly due to a proton
gradient; therefore, the presence of the proton un-coupler FCCP
in experiments A and D (Table 1) lowers the membrane potential
to a negligible value. On the other hand, a non zero membrane
potential should only be taken into account when analyzing the
results of experiments B and C, whose data are not used here to
demonstrate that CARD = CARA. In other words, the presence of
a non zero membrane potential, that would require the addition of
a term in equation (12), does not affect experiments A and D.
Consequently it would have no effect on eq (12) and on our whole
reasoning.
For testing the hypothesis that PfCRT is a carrier, corresponding to cells C2–5,5 in Figure 1, we need to consider the case of the
CQ* concentration inside the vacuole being lower (or not) than the
threshold value tPfCRT, i.e. being insufficient (or sufficient) to
saturate the carrier (hypotheses J2a and J2b).
In the first case, corresponding to cells C3–5,5 in Figure 1, the
outward flux is a linear function of the concentration of the
Test of hypotheses J1 (PfCRT is a channel) and J2 (PfCRT
is a carrier) [cells C1–5,5 Figure 1 and Figure 2B]. Let us
now consider the data obtained for resistant strains (C and D in
Table 1). We can use the values of pHDV and a to calculate the
concentration of the un-protonated chloroquine inside the vacuole
for CQR strains in the absence of FCCP (experiment C Table 1)
using equation (11). We call this value [CQ]DV,C to indicate that it
is related to experiment C and use eq (1) to calculate the outward
flux due to PfCRT, i.e.
JPfCRT ~Pcq ½CQe ½CQDV,C
obtaining ½CQDV,C ~1:53 10{12 M and JPfCRT ~2:799 10{3
nM cm=sec.
Next, we examine the specific hypotheses for PfCRT.
If PfCRT is a channel and the DV membrane potential is zero,
the transport is only driven by the difference in concentration of
the transported chloroquine form.
As a paradigm of our procedure, we describe the case where the
transported form is CQ+. A similar reasoning can be applied if the
transported species is CQ++ or a combination of CQ+ and CQ++.
We have
JPfCRT ~f ½CQz DV {½CQz e
ð12Þ
The function f(x) is increasing and it is zero when its argument is
zero. The presence of FCCP in experiment D (Table 1) implies
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Chloroquine Resistance
rewrite the expression for CAR as follows:
transported chloroquine form CQ*, i.e.
JPfCRT ~l ½CQDV
ð13Þ
CAR~
As shown in Text S1, section S8, the CARD values for each of the
chloroquine forms can be computed from eqs. (1), (11) and (13). In
particular:
where [CQTOT]x (with x being either DV or e) is the sum of the
three free chloroquine forms, namely [CQTOT]x = [CQ]x+[CQ+]x+
[CQ++]x = [CQ]x (1+[H+]x/k9+[H+]2x/k0). Note that equation (15) is a
general expression for CAR that, in the hypothesis H1, reduces to (11).
The value of [CQ:HM]DV,A can now be calculated using the
experimental value of CARA, while pHDV,B can be obtained from
equation (15) (where the dependence on pHDV is embedded in the
[CQTOT]DV term). The only positive solution admitted by the
equation is pHDV,B = 5.36, which is consistent with the experimental
data (Figure 2B and Table 3). This finding, obtained without making
any assumption on the nature of PfCRT, implies that hypothesis H2 is
plausible, regardless of the chloroquine species involved in the
chloroquine:HM binding and of the mechanism of action of the
mutated PfCRT. Consequently, none of the 20 cells C1–5,1–4 can be
shaded at this stage.
CARD ~204 for JPfCRT ~l ½CQDV
CARD ~684 for JPfCRT ~l ½CQz DV
CARD ~689
for JPfCRT ~l ½CQzz DV
Corresponding to cells C3–5,5 in Figure 1, respectively. The only
cases where the results are consistent with the data reported in
Table 1 are that PfCRT is an active carrier for either CQ+ or
CQ++ in a linear regime (Figure 2B). This implies that only cell
C3,5 is shaded in Figure 1 and Figure 2B.
A similar line of reasoning allows the CARD value to be
computed in the hypothesis that the CQ* concentration inside the
vacuole is not lower than the threshold value tPfCRT. In this case
(corresponding to cell C2,5 in Figure 1) we have
JPfCRT ~constant
Test of hypotheses J1 (PfCRT is a channel) and J2 (PfCRT
is a carrier) [cells C1–5,1–4 Figure 1]. As shown before, if
experiment A has been performed in saturation conditions, the
same is true for experiment B. We now need to infer what are the
conditions of experiment C, i.e. whether
½CQ : HMDV,A ~½CQ : HMDV,C
ð14Þ
ð16Þ
Notice that in experiment A there is no effect of PfCRT (being this
the case of a sensitive strain) or of pH differences (because of the
presence of FCCP), therefore all the chloroquine accumulation is
due to the chloroquine bound to HM. In particular, it is apparent
from eq. (15) that, without the [CQ:HM]DV contribution, CARA
would be 1. Equation (16) implies that the [CQ:HM]DV
contribution to CARC is equal to the [CQ:HM]DV contribution
to CARA hence, due to the additional contribution of pH to
chloroquine accumulation in C, we would have CARC.CARA.
Since the experimental data show that CARC,CARA, we can
reject the hypothesis represented by equation (16) and conclude
that the concentration of chloroquine in experiment C is not in the
saturation region (detailed numeric calculation can be found in
Text S1, section S9). In conclusion, if experiment A was
performed at saturating chloroquine:HM concentration, the
same holds for experiment B, but not for experiment C. This
implies that, in experiment C, we have [CQ:HM]DV = f(CQ*),
where f(CQ*) is a monotonic increasing function of one of the
chloroquine species.
In the hypothesis that PfCRT is a channel, the reasoning is
identical to that of section 1.1.1 for cell C1,5 of Figure 1 and leads
to the same conclusions: we cannot exclude that PfCRT is a
channel (Figure 2B) therefore, cells C1,1–4 in Figure 1 are not
shaded. Notice that no hypothesis on the chloroquine:HM binding
is required in the reasoning of section 1.1.1
We now discuss the case when the transported form is CQ+, i.e.
PfCRT is an active carrier that transports CQ+ in the linear
regime (cell C4,1–4 Figure 1). A similar reasoning can be applied if
the transported species are CQ or CQ++; the detailed calculations
for these other cases are reported in Text S1 (sections S11 and
S12).
In the hypothesis that PfCRT transports CQ+ and that the
dependency of the JPfCRT flux is linear in the concentration of
vacuolar CQ+ (JPfCRT = l[CQ+]DV) we need to consider the
following cases for chloroquine-HM binding:
In particular, JPfCRT,C = JPfCRT.D where the suffix C and D refer to
the experiments shown in Table 1. Eq. (14) and the CQR
membrane balance equation (eq (1)) implies [CQ]DV,C =
[CQ]DV,D. By substituting the values for [CQ]DV,D and a in
equation (11) and remembering that pHDV = pHe in the presence
of FCCP, we obtain CARD = 204. This value significantly differs
from the experimental one indicating that hypothesis J2b is not
compatible with the data (Figure 2B). Accordingly, the corresponding cell C2,5 in Figure 1 and Figure 2B is shaded.
Test of the hypothesis H2 ([CQ:HM]DV = constant). We
first use the values of CARA and CARB to infer the pHDV value
and verify its consistency with the experimentally known one.
Notice that, since CARA and CARB refer to CQS strains, we are
not initially making assumptions on PfCRT.
Let us test the possibility that [CQ*] is above the concentration
tHM,CQ needed to saturate the HM binding sites in the experiment
with sensitive strains in the presence of FCCP (Experiment A in
Table 1). As a first step, we need to understand whether this is the
case also for the experiment performed on sensitive strains in the
absence of FCCP (Experiment B in Table 1).
Because of the presence of FCCP in experiment A, we have:
pHA wpHB
and, consequently, [CQ]DV,A = [CQ]DV,B, [CQ+]DV,A,[CQ+]DV,B
and [CQ++]DV,A,[CQ++]DV,B, which implies that, in B, the
concentration of CQ* is either the same as in A or higher and,
therefore, [CQ:HM]DV,B must be in the saturation regime, where
[CQ:HM]DV = constant, as well. It follows that:
½CQ : HMDV,A ~½CQ : HMDV,B
Similarly to the linear regime hypothesis for HM binding
(hypothesis H1), it is possible to use equations (2), (3), and (4) to
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Ve
VDV ½CQTOT DV
VDV ½CQ : HMDV
z
ð15Þ
z
ð15Þ
Ve zVDV ½CQTOT e (Ve zVDV ) ½CQTOT e (Ve zVDV )
7
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Chloroquine Resistance
½CQ : HMDV ~f ½CQ
ð17aÞ
½CQ : HMDV ~f ½CQz
ð17bÞ
½CQ : HMDV ~f ½CQzz
ð17cÞ
½CQ : HMDV ~f CQTOT
ð17dÞ
We tested how stable our model is with respect to reasonable
variations of the CAR values. We repeated the whole procedure
using values of CAR derived for other parasite lines and for
variation within the experimental error obtaining the same
conclusions (data not shown).
In conclusion, ten of the forty cells of Figure 1, each
corresponding to a different assumption about the mode of
binding of chloroquine to HM in conjunction with a specific form
of PfCRT, are not shaded, i.e. they correspond in principle to
hypotheses consistent with the experimental data.
Sequence and structure analysis of PfCRT
Previous bioinformatics analyses of the PfCRT protein were
devoted at identifying the functional role of the PfCRT protein,
leading to different conclusions. On one hand several authors
[14,16] assigned PfCRT to the drug/metabolite transporter
(DMT) superfamily and, among the previously defined protein
families, reported that PfCRT has the highest similarity with the
drug/metabolite exporter (DME) family. On this basis, the authors
concluded that PfCRT is likely to function as an exporter of
metabolites in symport with H+.
Other studies proposed that PfCRT might share significant
sequence similarity with the ClC chloride channels of other
organisms [29,35], which would reinforce the hypothesis that the
protein acts as a gated aqueous pore.
As already mentioned by other authors [14], data supporting
the ClC similarity hypothesis are not available and were
impossible to reproduce using any available sensitive and updated
sequence analysis tool. On the contrary, as detailed later, several
different and effective methods strongly support the hypothesis
that PfCRT is an active carrier.
The HHsearch tool [39,40] identifies three protein families in
the Pfam database [41] with the highest similarity to PfCRT:
PF06027 (DUF914, E-value 1.2610234), PF08449 (UAA transporters, E-value 5.6610224), PF04142 (nucleotide-sugar transporters, E-value 7.8610223). These all belong to the Drug/
Metabolite Transporter clan CL0184, which consists of several
families of different secondary carriers, among which drug, sugar
and nucleotide antiporters have the highest similarity with PfCRT.
The alignment of PfCRT homologous with the closest Pfam family
(DUF914) is shown in Figure 3.
Two amino acids of PfCRT are known to be involved in
chloroquine resistance, 163 and 76, and therefore expected to be
involved in the transport mechanism. To verify whether this is
indeed the case, we built a homology model of the protein.
By searching the PDB database [42] with HHsearch, we
retrieved two significant hits (e-value,0.1). The significant
matches correspond to two solved structures (pdb codes 3B5D
and 2I68) of the same protein, EmrE. This protein is a small E.coli
multidrug transporter belonging to the same drug/metabolite
transporter family described above that acts by exchanging various
positively charged aromatic drugs across the plasma membrane
with protons. EmrE is a homodimer, each monomer being
composed by four membrane spanning helices. Two different
regions of PfCRT sequence match three helices of each EmrE
monomer, consistently with the presence of an internal sequence
symmetry in PfCRT. We also searched for homologous proteins
spanning the complete sequence of the protein to use as templates.
Three different and sensitive methods (Shrimp [43], Phyre [44]
and PROCAIN [45]) all identified the Glycerol-3-phosphate
transporter GlpT from E.Coli (PDB code 1PW4) as a homolog of
known structure with a significant sequence similarity spanning the
whole sequence (e-value,1024 in all cases). The protein was
ranked first in the searches performed with Shrimp and
corresponding to cells C4,1–4 of Figure 1, respectively.
Equation (15) clearly shows that the difference between CARC
and CARD is due to its second and third terms. Combining the
expression for the PfCRT flux JPfCRT = l([CQ+]DV) with that of
the membrane equilibrium (eq. (1)) we have that [CQ]DV =
[CQ]e/(1+[H+]DV l/(k9 Pcq) ), and hence
½CQTOT DV ~
l½H z DV
½CQe 1z
Pcq k’
{1
½H z DV ½H z 2DV
z
1z
k’
k’’
!
ð18Þ
that is, for any value of the unknown parameter l, an increasing
function of [H+]DV in the interval 0,pHDV,9.15, which is the
interesting one for plasmodium metabolism (the proof is reported
in Text S1, section S10). Being [H+]DV,C.[H+]DV,D, we have
[CQTOT]DV,C.[CQTOT]DV,D; therefore the second member of
equation (15) is larger for case C than for case D. As far as the
third member of the equation is concerned, we have to distinguish
the four cases (17a–d). Detailed calculations relating to each single
hypothesis for the chloroquine:HM binding (cases (17a–d)) are
reported in Text S1, section S13. Taking into account that
[H+]DV,C.[H+]DV,D and that f(CQ*) is monotonic, we eventually
obtain (Figure 2B):
Case 17a : ½CQ : HMDV,C v½CQ : HMDV,D
CARD ?CARC
Case 17b : ½CQ : HMDV,C w½CQ : HMDV,D
CARC wCARD
Case 17c : ½CQ : HMDV,C w½CQ : HMDV,D
CARC wCARD
Case 17d : ½CQ : HMDV,C w½CQ : HMDV,D
CARC wCARD
While in the first case no conclusion can be drawn on the
relationship between CARC and CARD (indicated by a question
mark in Figure 2B), cases (17b), (17c) and (17d) can be excluded
and the corresponding cells C4,2, C4,3 and C4,4 can be shaded in
Figure 1 and Figure 2B.
If JPfCRT = constant (cells C2,1–4 in Figure 1), this expression can
be used in the vacuolar membrane balance equation for CQR
strains (eq. (1)). Following the reasoning reported in Text S1,
section S14, we obtain that CARD,CARC, which is inconsistent
with the experimental data (Figure 2B). This implies that
hypothesis H2 in conjunction with the hypothesis that PfCRT is
a saturated chloroquine carrier is not plausible. Accordingly, cells
C2,1–4 (Figure 1 and Figure 2B) are shaded.
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Chloroquine Resistance
Figure 3. Alignment between the PfCRT family and the DUF914 family. Profile-profile alignment of PfCRT homologs (see Materials and
Methods) and the Pfam family DUF914, a member of the DMY clan. Residues are colored according to their physico-chemical properties
(green = hydrophobic, red = negatively charged, blue = positively charged, pink = polar, white = small, yellow = cysteine, gray = proline, orange = histidine). The image was generated using the MPI Bioinformatics Toolkit [52].
doi:10.1371/journal.pone.0014064.g003
In conclusion, sequence analysis strongly suggests that PfCRT is
an active carrier belonging to the Drug/Metabolite transporter
superfamily and, furthermore, a three-dimensional model based
on the basis of this evolutionary relationship is consistent with the
experimental data on the protein.
PROCAIN and second by Phyre. The top ranking protein
identified by Phyre (LacY) has the same fold and belongs to the
same superfamily as the Glycerol-3-phosphate transporter.
GlpT was therefore selected as the template for the model. It is
an active transporter presenting a 12-helix trans-membrane helix
protein with an internal sequence symmetry.
The final model (shown in Figure 4) was generated according to
the target/template alignment shown in Figure 5. The model
coordinates can be downloaded from the PMDB database [46] (Id
PM0076214).
Mapping of residues 163 and 76 in the three-dimensional model
of the protein (Figure 4) shows that they face each other and line
the path of the transported molecule. This is consistent with the
experimental observations and it appears plausible that the K76T
and S163R mutations can permit positive charged species to be
transported or not, respectively.
Discussion
The ongoing debate on PfCRT, the molecule responsible for
chloroquine resistance in Plasmodium, has not yet provided a
conclusive answer to the question of whether PfCRT is a channel
or a carrier.
The qualitative analysis of chloroquine accumulation, transstimulation and chloroquine efflux data has been used to support
both the channel and the carrier hypotheses by different authors
[26,27,29,30,34]. This is not surprising since we can analytically
demonstrate that, at least as far as the choloroquine accumulation
ratio experiments are concerned, the data are consistent with both
hypotheses. A similarly rigorous approach cannot be used for the
other experiment types since the problem is underdetermined.
This notwithstanding, we are able to show here that, if PfCRT is
a carrier, it can only transport protonated or di-protonated
chloroquine molecules and that choloroquine can either bind
heme or heme related species in the digestive vacuole regardless of
its charge or in its neutral form.
On the other hand, a reassessment of the evolutionary
relationships of the protein with state-of-the-art methods and
updated databases strongly suggests that the protein is indeed a
member of the Drug/Metabolite transporter clan. This conclusion
is further substantiated by the observation that a model based on
this detected evolutionary relationship is consistent with experimental data.
Taking together the results of these two interdisciplinary
approaches allow us to conclude that the chloroquine species are
transported out of the vacuole through PfCRT in their protonated
form, in agreement with studies such those presented by Lehane et
al [24,47] who provided evidence that the presence of chloroquine
increases the leak of H+ from the vacuole.
We would like to emphasize that our analytical approach does
not require ad-hoc hypotheses as it would be the case if we were to
model data coming from trans-stimulation and chloroquine efflux
experiments where parameters such as the rate of the vacuole pH
equilibration during chloroquine uptake or the kinetic constants of
chloroquine-hemozoine binding are unknown. On the other hand,
the model can be used effectively to interpret the results of
stationary experiments. As an example, recently Martin and
collaborators [25] set up a system in which PfCRT is expressed at
the surface of Xenopus leavis oocytes and measured the chloroquine
uptake which, in this system, is not influenced by chloroquine-HM
binding. The available data have been obtained in the prestationary state, but the same system could in principle be used to
measure chloroquine uptake at equilibrium in a pH-controlled
experiment. In this case a simplified version of our analytical
model could be employed to derive the systems parameters and
provide more detailed information about PfCRT.
Remarkably, our analysis conclusively demonstrates that
experimental data on the chloroquine accumulation ratio at
equilibrium are consistent with both hypotheses that the mutant
Figure 4. The model of the transmembrane region of PfCRT.
Residues K76 (left side) and S163 (right side) are shown in red using a
stick representation.
doi:10.1371/journal.pone.0014064.g004
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Chloroquine Resistance
Figure 5. Sequence alignment between the target PfCRT protein sequence and the sequence of the template (1PW4) as obtained by
Phyre.
doi:10.1371/journal.pone.0014064.g005
molecule is an active or a passive carrier of the drug and therefore
insufficient to distinguish between the two mechanisms, while at
the same time they can be used to restrict hypotheses on the nature
of the transported and HM-binding species in the two cases.
Our computational analysis of the sequence and a structural
bioinformatics approach strongly suggests that the mutated protein
acts as an active carrier of chloroquine and, in this assumption, we
can conclude that the PfCRT mutated protein confers resistance
by carrying either the mono or di-protonated chloroquine out of
the vacuole. It is tempting to speculate that the mechanism is that
the mutated PfCRT uses the H+ gradient to expel the protonated
form of chloroquine from the vacuole. Cationic transport
inhibitors could be tested to further support this hypothesis and,
perhaps, as starting point for developing novel therapies against
resistant malaria strains.
We hope that the example of the power of systems and
computational biology analysis of the data presented here will
convince the Plasmodium community to take advantage of our
results.
the cases, a statistically significant similarity of the PfCRT
sequence with that of the Glycerol-3-Phosphate transporter GlpT
from E.Coli (PDB code 1PW4) spanning the whole sequence of
both target and template proteins was detected.
Supporting Information
Text S1 Details of the calculations presented in the main
manuscript.
Found at: doi:10.1371/journal.pone.0014064.s001 (1.24 MB
PDF)
Table S1 HHpred results on Pfam and PDB databases.
Found at: doi:10.1371/journal.pone.0014064.s002 (0.02 MB
PDF)
Table S2 Phyre results.
Found at: doi:10.1371/journal.pone.0014064.s003 (0.02 MB
PDF)
Table S3 PROCAIN results.
Found at: doi:10.1371/journal.pone.0014064.s004 (0.02 MB
PDF)
Materials and Methods
Ethics Statement: N/A
Table S4 Shrimp results.
Found at: doi:10.1371/journal.pone.0014064.s005 (0.03 MB
PDF)
We performed sequence similarity searches on the nr database
[48] release of October 4th, 2010 using three iterations of CS-Blast
[49]. We selected the first 500 hits, all having an e-value lower
than 1e-5, and realigned their sequences using the multiple
sequence alignment tool MUSCLE [50]. Such alignment was then
used to scan the Pfam [41] and PDB [42] databases on October
5th, 2010 with the Hidden Markov Model based search method
HHpred [39], the results of which are reported in Table S1. Three
distant homology recognition tools, Phyre [51], Shrimp [43] and
PROCAIN [45], were used to identify templates spanning the
complete PfCRT sequence (see Table S2, Table S3 and S4). In all
Acknowledgments
We are grateful to Drs. Giombini and Raimondo for useful discussions.
Author Contributions
Conceived and designed the experiments: MC AV PM AT. Analyzed the
data: MC AV PM. Wrote the paper: AV AT.
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November 2010 | Volume 5 | Issue 11 | e14064