Download The Malaria Parasite`s Chloroquine Resistance Transporter is a

The Malaria Parasite’s Chloroquine Resistance Transporter is a
Member of the Drug/Metabolite Transporter Superfamily
Rowena E. Martin and Kiaran Kirk
School of Biochemistry and Molecular Biology, Faculty of Science, The Australian National University, Canberra, Australia
The malaria parasite’s chloroquine resistance transporter (CRT) is an integral membrane protein localized to the
parasite’s acidic digestive vacuole. The function of CRT is not known and the protein was originally described as
a transporter simply because it possesses 10 transmembrane domains. In wild-type (chloroquine-sensitive) parasites,
chloroquine accumulates to high concentrations within the digestive vacuole and it is through interactions in this
compartment that it exerts its antimalarial effect. Mutations in CRT can cause a decreased intravacuolar concentration of
chloroquine and thereby confer chloroquine resistance. However, the mechanism by which they do so is not understood.
In this paper we present the results of a detailed bioinformatic analysis that reveals that CRT is a member of a previously
undefined family of proteins, falling within the drug/metabolite transporter superfamily. Comparisons between CRT and
other members of the superfamily provide insight into the possible role of the protein and into the significance of the
mutations associated with the chloroquine resistance phenotype. The protein is predicted to function as a dimer and to be
oriented with its termini in the parasite cytosol. The key chloroquine-resistance-conferring mutation (K76T) is localized
in a region of the protein implicated in substrate selectivity. The mutation is predicted to alter the selectivity of the protein
such that it is able to transport the cationic (protonated) form of chloroquine down its steep concentration gradient, out of
the acidic vacuole, and therefore away from its site of action.
Introduction
The emergence and spread of malaria parasites that
are resistant to the widely-used antimalarial drug chloroquine (CQ) has been a disaster for world health. CQ is
a weak base that accumulates in the parasite’s digestive
vacuole, a lysosomal compartment in which haemoglobin,
taken up from the host cell cytosol via an endocytotic
feeding mechanism, is degraded to its component peptides
and haem. Within the vacuole CQ interferes with the
mechanism by which the potentially toxic haem monomers
are converted to the inert crystalline substance haemozoin,
causing monomeric haem to accumulate to levels that kill
the parasite.
Two proteins—Pgh1 (P-glycoprotein homolog 1;
Reed et al. 2000) and CRT (CQ resistance transporter;
Fidock et al. 2000)—have been implicated as playing
a role in CQ resistance. Both are integral membrane
proteins, localized to the parasite’s digestive vacuole
membrane. In Plasmodium falciparum, the most virulent
of the human malaria parasites, mutations in the CRT
protein (PfCRT) confer CQ resistance on otherwise
sensitive parasite strains (Sidhu, Verdier-Pinard, and
Fidock 2002). CQ-resistant parasites have a markedly
reduced concentration of CQ in their digestive vacuole
(Fitch 1970; Saliba, Folb, and Smith 1998); however,
neither the mechanism by which PfCRT influences the
intravacuolar concentration of the drug nor the normal
physiological role of this protein are understood.
In their original description of the protein, Fidock et al.
(2000) described PfCRT, together with orthologs from other
Plasmodium species and a more distant homolog from the
slime mould Dictyostelium discoideum, as being putative
channels or transporters containing 10 transmembrane
domains (TMDs). Very recently, two preliminary reports
Key words: malaria, Plasmodium falciparum, chloroquine, drug
resistance, PfCRT, drug/metabolite transporter.
E-mail: [email protected].
Mol. Biol. Evol. 21(10):1938–1949. 2004
doi:10.1093/molbev/msh205
Advance Access publication July 7, 2004
have assigned the PfCRT protein to the drug/metabolite
transporter superfamily (Martin, Trueman, and Kirk 2003;
Tran and Saier 2004). Here we present a detailed bioinformatic analysis of the protein and of the family and
superfamily to which it belongs. Comparisons between
PfCRT and members of the superfamily provide insight into
the possible role of the protein and into the significance of
the mutations associated with the CQ resistance phenotype.
Materials and Methods
Identifying Homologs of PfCRT
The amino acid sequence of PfCRT was queried
against the NCBI nonredundant protein database using
a BlastP (Altschul et al. 1990) search in which the query
sequence was not masked in areas of low compositional
complexity. This option was chosen because the filter
sometimes excludes from the analysis stretches of hydrophobic residues that correspond to the membrane-spanning
regions of the protein and which are therefore of biological
interest. Proteins retrieved by this search but excluded from
the subsequent phylogenetic study included duplicate
sequences and truncated proteins. Several proteins displayed areas of reasonable alignment with PfCRT (as
appraised by eye), even though the Blast output suggested
that the probability of homology was low (P . 1024).
A detailed bioinformatic analysis was performed on such
proteins; for each, the number and position of putative
TMDs was used to assess whether the regions of sequence
similarity corresponded to regions of alignment between
the predicted secondary structures of the retrieved protein
and PfCRT. Proteins that satisfied this criteria were then
queried against the NCBI nonredundant protein database
(using BlastP) and the Entrez Conserved Domain Database
(using Reverse Position-Specific Blast; Marchler-Bauer et
al. 2002) to determine their relationships to other proteins.
PfCRT was also queried against the Entrez Conserved
Domain Database as well as the Conserved Domain
Architecture Retrieval Tool database (Geer et al. 2002)
and was the subject of a Position-Specific Iterated Blast
Molecular Biology and Evolution vol. 21 no. 10 Ó Society for Molecular Biology and Evolution 2004; all rights reserved.
The Chloroquine Resistance Transporter 1939
Phylogenetic Analyses
Regions of the alignment that could not be aligned
unambiguously were excluded prior to analysis. A
phylogenetic tree was estimated using the NeighborJoining method (Saitou and Nei 1987) and uncorrected
(‘‘p’’) amino acid distances in MacVectorÔ 7.1. Ties in the
tree were resolved randomly and a bootstrap analysis
(Felsenstein 1985) was performed with 1,000 replicates.
FIG. 1.—Neighbour-Joining tree of CRT family based on uncorrected distances. Numbers indicate the percentage of 1,000 bootstrap
replicates which support the topology shown. The scale bar represents the
number of substitutions per site for a unit branch length.
(Altschul et al. 1997) search of the NCBI nonredundant
protein database.
Alignment of Hydropathy Profiles
Hydropathy profile alignments were generated at
http://bioinformatics.weizmann.ac.il/hydroph/ using KyteDoolittle (x 2 1) values, a window size of 17, and an
algorithm that introduces gaps into the alignment to find
the best match between two profiles. The final alignment
of the hydropathy plots was compiled and edited in
AdobeÒ PhotoshopÒ 6.0.1.
Secondary Structure Predictions
TMpred (www.ch.embnet.org/software/TMPRED_
form.html) and TMMHM (www.cbs.dtu.dk/services/
TMHMM-2.0/) were used to detect putative membranespanning domains in the sequences of the proteins of
interest. Predictions of protein orientation were made on
the basis of the ‘positive inside’ rule (von Heijne 1986;
van Klompenburg et al. 1997) as well as by TMMHM
(which incorporates the positive inside rule in its prediction of membrane protein topology [Sonnhammer, von
Heijne, and Krogh 1998]). The predicted secondary
structure of loop 7 of the CRT family proteins was
obtained using the PredictProtein server (http://cubic.
bioc.columbia.edu/predictprotein/).
Construction of Alignments
The ClustalW program (Thompson, Higgins, and
Gibson 1994) in MacVectorÔ 7.1 was used to generate
and edit alignments. Sequences for various DMT proteins
have been described (e.g., Jack, Yang, and Saier 2001),
and these were used to retrieve many DMT members from
the NCBI database. Proteins of different DMT families
have diverged considerably at the amino acid level and this
can make a one-step alignment method error-prone. We
therefore first aligned proteins within a family and then
used the ClustalW profile-alignment tool to assemble the
families into one large alignment. This alignment corresponded very well with the alignment of the predicted
TMDs in these proteins. The first half of the DMT
superfamily alignment was aligned to the second using the
ClustalW profile alignment tool.
Results and Discussion
PfCRT Belongs to a Family of Proteins
The proteins with the greatest similarity to PfCRT are
homologs from other Plasmodia species (P. vivax, P.
knowlesi, P. yoelii yoelii, P. chabaudi, and P. berghei) and
these are retrieved by a BlastP search with P values in the
range of 102162 to 102137. The P value is the probability
that the sequence similarity shared by the query protein
and the retrieved protein arose by chance. A P value
,1024 is considered to indicate a significant sequence
similarity between the two proteins, consistent with their
having a related biological function. Following the
Plasmodia proteins, the next best hits against PfCRT
include a protein from Cryptosporidium parvum (1 3
10225), the D. discoideum protein (4 3 10220), and several proteins from Arabidopsis thaliana (2 3 1025). A
phylogenetic analysis performed on the sequence alignment of the Plasmodia, C. parvum, D. discoideum, and
A. thaliana proteins (fig. 1) provides good evidence in
support of the hypothesis that these proteins form a family.
The CRT Proteins Are Related to Known Transporters
The search for relatives of PfCRT in the NCBI database
retrieved many proteins that had P values . 1024, indicating low, if any, homology to PfCRT. Nevertheless, several
of these proteins showed a reasonable similarity in sequence
to PfCRT over specific regions of the alignment. All such
proteins are members of the same group of transport
proteins, the drug/metabolite transporter (DMT) superfamily.
When the D. discoideum, C. parvum, and A. thaliana
proteins were queried against the NCBI database, they also
retrieved many proven or putative transporters of the DMT
superfamily. Three iterations of a Position-Specific Iterated
Blast search of the NCBI database using PfCRT as the query
sequence retrieved, with good significance, several characterized transporters of the DMT superfamily. Proteins of the
same family typically share distinct modules or domains that
have a common evolutionary origin and function. The
PfCRT protein was queried against the Entrez Conserved
Domain Database using Reverse Position-Specific Blast and
was found to have weak, but significant, hits to DMT
superfamily conserved domains. These observations are
consistent with the hypothesis that the proteins of the CRT
family form part of, or are related to, the DMT superfamily.
Members of the CRT and DMT Families Have
Similar Hydropathy Plots
A simple and commonly used predictor of the
structure of a membrane protein is the hydropathy plot,
1940 Martin and Kirk
FIG. 2.—An alignment of the hydropathy plots for the P. falciparum (yellow), D. discoideum (red) and A. thaliana (green) members of the CRT
family and for the E. coli YdeD amino acid effluxer (blue) of the DMT superfamily. The positions of the predicted 10 TMDs are indicated by white
bars. Gaps in the alignment are indicated below the plot by appropriately colored lines.
in which putative TMDs and the connecting hydrophilic,
extra-membrane loops are detected as ‘peaks’ and
‘troughs,’ respectively, in a plot of the hydrophobicity
index of the polypeptide. Transporters of the same family
usually share a very similar hydropathy plot, even when the
underlying polypeptide sequences have diverged considerably (Lolkema and Slotboom 1998). Members of the DMT
superfamily, like those of the CRT family, are predicted to
contain 10 TMDs. Figure 2 shows an alignment of the
hydropathy plots for PfCRT (yellow line), the D. discoideum protein (red line), one of the A. thaliana proteins
(green line), and a protein from the DMT superfamily (the
E. coli YdeD amino acid effluxer; blue line), consistent with
these proteins having similar structure. In pairwise
sequence alignments between members of the CRT family
and the DMT protein, the regions of sequence similarity
were found to correspond to the regions of alignment of
predicted TMDs in the two proteins.
et al. 2003). The NST and TPT families clustered together
on a branch separate from the DME proteins and the DME
family was comprised of many nodes, one of the outermost
being the plant DME proteins. The RhaT, GRP, and CEO
families clustered together and are distantly related to the
DME proteins. The CRT family placed within the DMT
superfamily, in which it branched from the DME proteins,
after the RhaT, GRP, and CEO cluster had diverged.
Bootstrap analysis was performed on a reduced data
set of 53 proteins that included representatives from the
major groups within the DMT superfamily. As shown in
figure 3, the bootstrap values support the placement of the
CRT proteins within the DMT superfamily, where they
cluster as a distinct family that branches between the DME
and NST families. The analysis was repeated on another
two subsets of ;50 proteins drawn from the full data set of
368 proteins and both yielded results similar to the tree
presented in figure 3.
The CRT Family Falls Within the DMT Superfamily
The CRT Proteins Arose from a Gene Duplication Event
The DMT superfamily consists of a number of
discrete membrane protein families including the drug/
metabolite effluxer (DME) family, the L-Rhamnose
symporter (RhaT) family, the glucose/ribose permease
(GRP) family, the C. elegans ORF (CEO) family (no
members of which have been characterized as yet), the
nucleotide-sugar transporter (NST) families, the triosephosphate transporter (TPT) family and the plant organocation permease (POP) family (Jack, Yang, and Saier
2001). An alignment of 368 proteins belonging to the
DMT superfamily and CRT family was constructed from
10 CRT, 206 DME, 6 RhaT, 12 GRP, 7 CEO, 76 NST, 41
TPT, and 10 POP proteins. Four transporters from each of
three unrelated 10-TMD transporter families—the
Ca21:cation antiporters (CaCA), the C4-dicarboxylate
importers (Dcu), and the glutamate:Na1 symporters
(ESS)—were included as outgroups.
The ‘best tree’ was estimated using the NeighborJoining method and the relationships within the superfamily
were found to be similar to those described previously
(Jack, Yang, and Saier 2001; Ward 2001; Knappe, Flugge,
and Fischer 2003; Livshits et al. 2003; Martinez-Duncker
Within members of the DMT superfamily there is
significant homology between the first half of the protein
(encompassing TMDs 1–5) and the second half (encompassing TMDs 6–10), consistent with these transporters
having arisen from an ancestral gene that underwent an
internal duplication event (Jack, Yang, and Saier 2001).
Figure 4 shows alignments of the two halves of
a representative selection of DMT proteins together with
three members of the CRT family (including PfCRT). The
first TMD is aligned with the sixth TMD, the second TMD
with the seventh, and so on. As well as there being
significant similarities along the whole lengths of the
different proteins, there are similarities between the two
halves of the proteins, including in the CRT proteins.
The Membrane Orientation of the CRT Proteins
As is evident from figure 4, there is a general trend
within the DMT superfamily (including the CRT proteins)
for the even-numbered extra-membrane loops to have
a greater proportion of positively charged residues than
the odd-numbered loops. The N- and C-termini also have
The Chloroquine Resistance Transporter 1941
FIG. 3.—Neighbour-Joining tree of the DMT superfamily based on uncorrected distances. The analysis included three members of the CRT family
and 38 DMT sequences. Four of the DMT proteins are characterized transporters of the nucleotide-sugar transporter (NST) family and 34 are known or
putative members of the drug/metabolite efflux (DME) family. Included for comparison were four sequences from each of three unrelated 10 TMD
transporter families: the Ca21:cation antiporters (CaCA), the C4-dicarboxylate importers (Dcu), and the Glutamate:Na1 symporters (ESS). Numbers
indicate the percentage of 1,000 bootstrap replicates which support the topology shown. The scale bar represents the number of substitutions per site for
a unit branch length.
!
FIG. 4.—The alignment of the first half (encompassing TMDs 1–5) with the second half (encompassing TMDs 6–10) of a representative selection
of DMT proteins, including three CRT family members. Residues are shaded as follows: positively charged, blue; negatively charged, red; tryptophan
and tyrosine, white; polar, proline, and glycine, grey; remaining nonpolar, yellow. The conserved proline in TMDs 4 and 9 is highlighted in green. The
variant N- and C-termini have been omitted. In some proteins loop 2 and/or 7 has been truncated and this is indicated by a solid black line. ‘(p)’
indicates that the assignment of function is only putative. The proteins are as follows: 1, M. musculus gi 2499227 (Eckhardt et al. 1996); 2,
D. melanogster gi 21355345 (Martinez-Duncker et al. 2003); 3, K. lactis gi 6016590 (Abeijon, Robbins, and Hirschberg 1996); 4, C. elegans gi
20140026 (Berninsone et al. 2001); 5, C. albicans gi 14971021 (Nishikawa et al. 2002); 6, H. sapiens gi 14009667 (Lubke et al. 2001; Luhn et al.
2001); 7, Z. mays gi 1352200 (Fischer et al. 1994); 8, A. thaliana gi 15219121; 9, E. coli gi 26107188 (Livshits et al. 2003); 10, E. coli gi 25367911
(Santiviago et al. 2002); 11, B. subtilis gi 1175623 (Jack, Yang, and Saier 2001); 12, P. agglomerans gi 4098977; 13, E. coli gi 26250715 (Jack, Yang,
and Saier 2001); 14, F. nucleatum gi 19705391; 15, M. loti gi 13473318; 16, M. rubra gi 2062658 (Berg, Hilbi, and Dimroth 1997); 17, E. coli gi
13361606 (Dassler et al. 2000); 18, T. erythraeum gi 23043464; 19, B. fungorum gi 22988226; 20, P. marinus gi 33862898; 21, T. fusca gi 23018393;
22, B. subtilis gi 16081133 (Jack, Yang, and Saier 2001); 23, C. aurantiacus gi 22972802; 24, A. thaliana gi 25408555; 25, A. thaliana gi 18415262;
26, A. thaliana gi 21536591; 27, P. falciparum gi 23612473 (Fidock et al. 2000); 28, D. discoideum gi 11139714; 29, S. xylosus gi 2226001 (Fiegler
et al. 1999); 30, C. elegans gi 13384556.
1942 Martin and Kirk
The Chloroquine Resistance Transporter 1943
FIG. 5.—Alignment of CRT proteins over TMD 1 and loop 7. Residues are shaded as follows: positively charged, black; negatively charged, dark
grey; tryptophan and tyrosine, white; polar, proline and glycine, mid-grey; remaining nonpolar, light grey. The position of the K76T mutation in TMD
1 is indicated (star) and CQ-resistant parasite strains (numbered 5 and 6) are highlighted.
a preponderance of positive charge. This makes it likely that
the even-numbered loops, together with the N- and Ctermini, are located at the cytoplasmic face of the membrane
(predicted by the positive inside rule [von Heijne 1986; van
Klompenburg et al. 1997] and by TMHMM [Sonnhammer,
von Heijne, and Krogh 1998]). Such an orientation (i.e.,
termini in the cytosol) has been proven experimentally for
two DMT proteins, the mouse CMP-sialic acid transporter
(NST family; Eckhardt, Gotza, and Gerardy-Schahn 1999)
and the PecM protein of Erwinia chrysanthemi (DME
family Rouanet and Nasser 2001).
For the RhaT, GRP, and CEO proteins it is the oddnumbered loops that have the higher proportion of positive
charge, suggestive of the opposite topology, and this has
been demonstrated experimentally for the S. typhimurium
RhaT protein (for which the termini were found to be
noncytosolic [Tate and Henderson 1993]).
7 is a relatively long hydrophilic domain, changes in which
have been shown to influence transporter activity. The
activity of the mouse CMP-sialic acid transporter is
reduced when an epitope is inserted into loop 7 (but not
loop 2), and increasing the length of the tag causes
a further reduction in transporter activity (Eckhardt, Gotza,
and Gerardy-Schahn 1999). As illustrated in figure 5, loop 7
is especially long and well conserved in composition in
the proteins of the CRT family, and the predicted structure
of this region (given by the PredictProtein sever) was
similar for each protein: a ‘compact globular domain’ that
is formed by a nine amino acid alpha helix followed by two
short beta sheets (although in some proteins the first beta
sheet may instead be an alpha-helix).
The predicted orientation of PfCRT in the membrane
(i.e., with the protein termini in the cytosol) places loop 7
in the digestive vacuole, where it may have a role in
modulating the activity of the transporter.
The Significance of the Extra-Membrane Loops
Having established that PfCRT is a member of the
DMT superfamily it is possible to assign putative functions
to different regions of the PfCRT protein on the basis
of previous studies of other members of the superfamily
(fig. 4).
A striking feature of figure 4 is the conservation of
structural elements throughout the superfamily, despite the
considerable divergence in sequence, mode of transport,
and substrate specificity. For instance, the length and
composition of the loops are conserved between proteins
from different families. Furthermore, the loop regions in
the second half of the transporter bear significant similarity
to the corresponding loops in the first half. Loops 3 and 8
and 4 and 9, are particularly well conserved. Studies with
proteins from both the NST and DME families have
revealed that the insertion of an epitope tag or reporter
molecule in loops 3, 4, 8, and 9 can inactivate the
transporter and in some instances cause the protein to be
localized incorrectly within the cell and/or degraded (Tate
and Henderson 1993; Eckhardt, Gotza, and GerardySchahn 1999; Rouanet and Nasser 2001). By contrast, the
same sequences introduced into loops 1, 2, 5, and 6 have
no effect on transporter activity or localization.
Compared to the other loops, 2 and 7 show less
sequence conservation and show significant variation in
length between DMT proteins. In most DMT proteins loop
The Presence of Helix Packing Motifs in
Transmembrane Domains 5 and 10 Indicates
that PfCRT May Function as a Dimer
While the structure of the DMT transporters has been
retained over time and evolution, the underlying amino
acid sequence has proven more plastic. Nevertheless, some
regions of sequence have been strongly conserved. In
TMDs 5 and 10 there are two conserved glycines that are
separated by six hydrophobic residues (fig. 4; Knappe,
Flugge, and Fischer 2003). This motif (GxxxxxxG) is
a common feature of membrane proteins, in which it is
thought to facilitate the packing of membrane-spanning
helices, leading to the association of TMDs to form
oligomers (Liu, Engelman, and Gerstein 2002). Other
small residues (alanine, serine, and threonine) can replace
one of the glycines in a glycine-packing motif (Russ and
Engelman 2000; Eilers et al. 2002) and such a substitution
has occurred in a number of the DMT proteins, including
PfCRT (fig. 4). TMDs 5 and 10 are known to have a role in
mediating the formation of homo-dimers by the NST and
TPT transporters (Abe, Hashimoto, and Yoda 1999; Ishida
et al. 1999; Streatfield et al. 1999; Gao and Dean 2000)
and nonconservative mutations within the putative packing
motifs in TMDs 5 and 10 abrogate transporter activity as
well as stability (Ishida et al. 1999; Streatfield et al. 1999).
Although it is not known whether members of the DME
1944 Martin and Kirk
family function as dimers, the conservation of the
GxxxxxxG motif in DME (as well as CRT proteins) are
consistent with their doing so.
Different Transmembrane Domains Are Implicated in
Substrate Recognition, Binding, and Translocation
Many studies with NST or TPT proteins have shown
that substitutions at conserved positions in TMDs 3, 4, 8,
and 9 interfere with the binding and translocation of the
substrate (a divalent anion). The mutations either impair
the rate of transport or abolish transport altogether, while
not affecting either the localization of the protein within
the cell or its ability to form homo-dimers (Abe,
Hashimoto, and Yoda 1999; Berninsone et al. 2001;
Gao, Nishikawa, and Dean 2001; Lubke et al. 2001; Luhn
et al. 2001; Oelmann, Stanley, and Gerardy-Schahn 2001;
Etzioni et al. 2002; Knappe, Flugge, and Fischer 2003).
A large proportion of these functionally important residues
are located in TMDs 4 or 9; in both cases they are
concentrated in the center of the helix where they form
a strongly conserved region of polar and positively
charged residues that is thought to constitute a substrate
binding motif (Fischer et al. 1994; Gao, Nishikawa, and
Dean 2001; Lubke et al. 2001).
The binding motif regions in TMDs 4 and 9 are also
well conserved in DME and CRT proteins, but they do not
contain positively charged residues. Instead, there is
a conserved proline, usually in both TMDs 4 and 9,
although a few proteins (including the Arabidopsis CRT
protein) have a proline in only one of the binding motifs
(fig. 4). The role of these conserved prolines in the
function of DME transporters has not been studied, but
proline residues located in the central part of a transmembrane helix are known to be essential for the biological
activity of a number of membrane transport proteins
(Webb, Rosenberg, and Cox 1992; Lin, Itokawa, and Uhl
2000; Shelden et al. 2001; Koike et al. 2004). The proline
ring distorts the normal structure of a membrane-spanning
helix via two mechanisms: a kink is introduced in the helix
backbone to avoid a steric clash with the proline ring, and
the hydrogen bonds that would normally stabilize this
region of the helix are unable to form (Woolfson and
Williams 1990; Visiers, Braunheim, and Weinstein 2000;
Cordes, Bright, and Sansom 2002). This results in a flexible
‘hinge’ point in the helix that has the capacity to form
hydrogen bonds (perhaps with a substrate). The presence
of a proline hinge in the putative binding motif of DME
and CRT proteins suggests a role for this residue in the
binding and translocation of substrates.
The codons encoding both proline and glycine are
GC-rich and, as such, these codons are the least likely to
be retained by chance in the AT-rich genomes of
Plasmodium and Dictyostelium (Stevens and Arkin
2000). This is consistent with there being a critical role
for the putative helix-packing glycine motifs and the intraTMD proline residues in the function of CRT proteins.
While discrete regions of the DMT proteins are
implicated in the binding and translocation of the substrate,
other elements of the transporters have been implicated in
the recognition of, and discrimination between, substrates.
The participation of residues from a number of TMDs in
substrate recognition is not uncommon among transporters. For example, in members of the major-facilitator
superfamily, eight out of the 12 TMDs are predicted to be
involved in determining the substrate specificity of the
transporter (Hirai et al. 2003). The functional analyses of
chimeras constructed from two human NSTs, the UDPgalactose transporter, and the CMP-sialic acid transporter,
revealed the participation of TMDs 1, 2, 3, 7, and 8 in determining substrate specificity (Aoki, Ishida, and Kawakita
2001, 2003). The involvement of TMDs 3 and 8 is not
surprising, as these domains are also thought to influence
the binding and translocation of the substrate; residues in
these helices are likely to face the translocation pore where
they may interact with the substrate.
Amino acid residues are considered to be potentially
‘substrate-specific’ when they are conserved among transporters with identical substrates, but are different between
transporters of differing substrate specificity. In DMT
proteins there are many such residues present in TMDs 2
and 7 (fig. 4). Indeed, TMD 7 is one of the helices that
varies the most in sequence between DMT proteins of
different subgroups. Consistent with this observation, the
substrate specificity of the UDP-galactose transporter is
broadened to include CMP-sialic acid simply by replacing
TMD 7 with the corresponding helix from the CMP-sialic
acid transporter. TMD 1 is also crucial for determining the
substrate specificity of hybrids between the UDP-galactose
and CMP-sialic acid transporters, and it is thought to have
a similar involvement in the substrate specificity of the
UDP-galactose transporter from fruit fly (Segawa, Kawakita, and Ishida 2002). Similarly, in TPT proteins, residues
in TMD 1 are proposed to line the translocation pore of the
transporter (Knappe, Flugge, and Fischer 2003).
TMDs 7, 8, 9, and 10 all fulfill the same role in
transporter function as the corresponding domains in the
first half of the protein (fig. 6). It might therefore be
expected that TMD 6, as the counterpart of TMD 1 in the
second half of the protein, should also play a role in
determining substrate specificity. Experimental support for
this comes from a study with the hamster CMP-sialic acid
transporter (Eckhardt, Gotza, and Gerardy-Schahn 1998).
Mutation of a glycine residue in TMD 6 to glutamate,
glutamine, or isoleucine severely reduces transporter
activity without affecting the expression or trafficking of
the protein. Overexpression of the mutant proteins restores
a low level of CMP-sialic acid transport, consistent with
the mutations having affected the affinity of the transporter
for its substrate.
The Chloroquine Resistance-Conferring K76 Mutation
Lies in a Region Implicated in Substrate Selectivity
The substitution of the lysine at position 76 for
threonine (K76T) has been identified as a crucial determinant of PfCRT-mediated CQ resistance (Sidhu,
Verdier-Pinard, and Fidock 2002). As depicted in figure 6,
this mutation lies towards the C-terminal end of TMD 1,
a region of the transporter that we predict to be involved in
substrate recognition. Field isolates from both Old and New
World strains of CQ-resistant parasites all have the critical
The Chloroquine Resistance Transporter 1945
FIG. 6.—Charge distribution, topology, and putative roles of the TMDs of PfCRT. Positively charged residues (lysine or arginine) predicted to lie
in the extra-membrane segments or in the termini regions are shown in black. The putative TMDs are numbered 1–10. The position of the CQresistance-associated K76T (TMD 1) and A220S (TMD 6) mutations (black), the conserved prolines (TMDs 4 and 9; dark grey) and the helix packing
motifs (GxxxxxxT; TMDs 5 and 10; mid-grey) are highlighted. TMDs 4 and 9 (boxed in dark grey) are implicated in the binding and translocation of
substrates by DMT proteins. The conserved helix packing motifs in TMDs 5 and 10 (boxed in mid-grey) play a role in the formation of homo-dimers by
TPT and NST proteins. Certain residues in TMDs 3 and 8 (boxed in light grey) in DMT transporters assist in the binding and translocation of the
substrate and both domains also influence the substrate specificity of the transporter. Several elements of the DMT transporter seem to be involved in
recognizing and discriminating between substrates. Along with TMDs 3 and 8, TMDs 1, 2, 6, and 7 (boxed in black) also participate in determining
substrate specificity. The topology cartoon was generated using the TOPO2 server (www.sacs.ucsf.edu/TOPO/topo.html).
K76T mutation, accompanied by a number of what are
thought to be ‘compensatory’ PfCRT mutations that enable
the transporter to maintain a semblance of its normal
physiological role (Carlton et al. 2001). The A. thaliana
CRT proteins, like the PfCRT proteins of CQ-sensitive
strains, have a positively charged residue in this position
(fig. 5). By contrast, the D. discoideum and C. parvum
proteins are more similar to the PfCRT proteins from
resistant strains of P. falciparum in that they have a serine
(serine and threonine are hydroxy amino acids and a S ! T
mutation is considered to be a conservative substitution).
A CQ-sensitive isolate from Sudan (106/1) contains
all of the PfCRT mutations found in CQ-resistant strains
from the Old World, with the exception of the K76T mutation which has reverted to the wild-type form (Fidock
et al. 2000). When this strain was subjected to CQ-selection
pressure in vitro, two CQ-resistant clones arose, both
with novel mutations in the K76 position (K76I and
K76N; Fidock et al. 2000; Cooper et al. 2002).
Characterization of these mutants revealed that the K76I
mutation had the unusual effect of increasing the parasite’s
sensitivity to quinine while decreasing its sensitivity to the
diastereomer quinidine. This adds further support to the
hypothesis that TMD 1 of PfCRT, and in particular
position 76, is involved in determining the substrate
specificity of the transporter. The absence of the K76I and
K76N mutations in CQ-resistant field populations (and the
prevalence of the K76T mutation) may reflect a less fit
phenotype in vivo for these artificially-derived mutants
(Cooper et al. 2002). This is consistent with the
observation that only two types of amino acid residues
are found in this position among other proteins of the CRT
family: positively charged or hydroxy.
Warhurst and colleagues (2002) have suggested
previously that PfCRT is related to proteins of the chloride
channel (ClC) family, members of which have been
determined to possess 18 alpha helices (Dutzler et al.
2002). In their analysis, the region of PfCRT containing
the K76T mutation was predicted to correspond to helix C
of the Salmonella typhimurium ClC protein and the loss of
the positive charge in this position was postulated to alter
the specificity of the parasite chloride channel, such that it
permitted the transport of chloroquine. However, residues
in helix C of the S. typhimurium ClC protein are not
known to influence substrate specificity, nor do they line
either the channel’s selectivity filter or translocation pore
(Dutzler et al. 2002). The mechanism by which the K76T
mutation would influence the selectivity of a channel of
this type, and thereby confer chloroquine resistance, is
therefore unclear.
Apart from the K76T mutation, there is another
PfCRT mutation (A220S) conserved in most CQ-resistant
isolates and absent from CQ-sensitive strains (with the
exception of the 106/1 strain). The A220S mutation is
located in TMD 6 and does not confer resistance in the
absence of the K76T mutation (e.g., 106/1). However, its
presence in most CQ-resistant strains analyzed to date
suggests that it acts in synergy with K76T, perhaps by
1946 Martin and Kirk
FIG. 7.—Model for the mechanism of PfCRT-mediated resistance to CQ. The protein is shown as a dimer, functioning to export ‘metabolites’
(perhaps amino acids or peptides, and perhaps in symport with H1) from the parasite’s digestive vacuole (DV). The ‘positive-inside’ rule, and a presumed
inwardly-positive electrical potential across the DV membrane, predicts the N- and C-termini to be cytosolic and the ‘compact globular domain’ of loop 7
to be located at the vacuolar face of the membrane. CQ is a diprotic weak base and therefore accumulates in the acidic DV in the protonated (positively
charged, CQ21) form. In parasites expressing ‘wild type’ PfCRT, the positive charge of K76 prevents the interaction of CQ21 with the transporter. The
CQ resistance–conferring K to T mutation removes the positive charge and alters the substrate selectivity, allowing CQ21 to interact with the transporter
and to be effluxed from the DV, perhaps in symport with H1. This results in a reduction of the concentration of CQ within the vacuole.
aiding the recognition of CQ as a substrate for PfCRT or as
a compensatory mutation that stabilizes the interaction of
the transporter with its physiological substrate(s). The
location of the A220 mutation in a PfCRT domain
predicted to participate in substrate recognition is consistent with both of these scenarios.
Substrates Effluxed by DME Transporters
The members of the DMT superfamily bearing the
closest similarity to the CRT proteins in the region of the
substrate binding motif fall within the DME transporter
subfamily. Substrates for DME transporters include amino
acids, weak bases, and organic cations. The YdeD protein
of E. coli exports cysteine metabolites (Dassler et al. 2000),
whereas the E. coli YbiF protein exports a broad range of
amino acids including homoserine, threonine, lysine, and
histidine (Livshits et al. 2003). In other species of bacteria,
DME proteins are implicated in the efflux of methylamine
(MttP; Ferguson and Krzycki 1997), the di-cationic
herbicide methyl viologen (YddG; Santiviago et al. 2002)
and the pigment indigoidine, which is, like chloroquine,
a weak base (PecM; Rouanet and Nasser 2001). The fact
that DME transporters are known to transport both weak
bases and divalent organic cations lends support to the
hypothesis that the CQ-resistant form of PfCRT transports
the chloroquine in the di-cationic form.
DME systems are postulated to be H1-coupled and
this has been confirmed experimentally for at least one
DME transporter (the E. coli YbiF protein) [Livshits et al.
1993].
The Role of PfCRT in Chloroquine Resistance
Figure 7 shows a model for the mechanism of
PfCRT-mediated CQ-resistance, based on the insights
gained from the bioinformatic analysis presented here, and
consistent with previous reports of enhanced CQ efflux
from CQ-resistant parasites (Krogstad et al. 1987;
Sanchez, Stein, and Lanzer 2003). The protein is shown
as a dimer, functioning to export ‘metabolites’ from the
parasite’s digestive vacuole. The facts that (1) a number of
related DME proteins transport amino acids, and (2) that
the only known metabolite transport function of the
digestive vacuole is the efflux of peptides (Kolakovich et
al. 1997) and perhaps amino acids, prompt the hypothesis
that PfCRT is an amino acid/peptide transporter (perhaps
H1-coupled), but this remains to be tested.
The protein is predicted, on the basis of the ‘positiveinside’ rule as well as by TMHMM, to be oriented with the
N- and C-termini in the parasite cytosol and the ‘compact
globular domain’ of loop 7 located at the vacuolar face of
the membrane. In this orientation, the crucial CQRconferring K76T mutation lies close to the surface of the
vacuolar face of the membrane, within a region of the
protein postulated to be involved in substrate selectivity.
The positive charge is predicted to play a key role in
repulsing the protonated (cationic) form of chloroquine
(CQ21, the predominant species present within the acidic
vacuole) and preventing it from interacting with the
transporter. The CQR-conferring mutation of Lys to Thr
(or Ile or Asn) removes the positive charge, allowing
CQ21 to interact with and be transported by the protein,
down the steep outward CQ21 concentration gradient
(again perhaps in symport with H1). On exiting the
vacuole and entering the parasite cytosol (pH 7.3; Saliba
and Kirk 1999), the CQ is deprotonated and diffuses out of
the parasite as the neutral (membrane-permeant) species.
The net result is a decrease in the overall concentration of
CQ at its site of action within the digestive vacuole, and
hence a decreased CQ-sensitivity of the parasite.
Experimental testing of the key aspects of the model
presented in figure 7 await the expression of PfCRT in
a heterologous system in a form in which the transport
properties of the protein can be investigated directly. PfCRT
has been successfully expressed in yeast (Zhang, Howard,
and Roepe 2002); however, there has not, as yet, been any
direct demonstration of its transport function. Efforts are
The Chloroquine Resistance Transporter 1947
presently underway to express the protein in Xenopus laevis
oocytes and to measure and compare the transport of
radiolabeled chloroquine via PfCRT with and without the
K76T mutation. It is predicted that oocytes expressing
PfCRT from CQ-resistant strains (i.e., having the K76T
mutation) in their plasma membrane will transport [3H]CQ,
whereas those expressing wild-type PfCRT from CQsensitive strains (having K76) will not. The successful
expression of PfCRT in Xenopus oocytes will also allow: (1)
a direct test of the hypothesis that PfCRT proteins from both
CQ-resistant and CQ-sensitive strains transport amino
acids/peptides; (2) screening of other classes of substrate
for their ability to be transported via PfCRT; (3) the
determination of whether PfCRT-mediated transport is H1
coupled; and (4) an investigation of whether, as has been
proposed (Warhurst 2003), the chloroquine resistance
reversal agent verapamil interacts directly with PfCRT (in
TMD 1) to inhibit the transport of CQ. Such experiments
have the potential to yield important insights into the
molecular mechanism underlying chloroquine resistance.
Acknowledgments
The authors are grateful to Stephen Allen and Jan
Martin for technical assistance and to Rhys Hayward,
Kevin Saliba, and John Trueman for helpful discussions.
This work was supported by the Australian National
Health and Medical Research Council (grant ID
179804).
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Michele Vendruscolo, Associate Editor
Accepted June 28, 2004