School of Biochemistry and Molecular Biology, Faculty of Science, The Australian National University, Canberra, Australia
Correspondence: E-mail: kiaran.kirk{at}anu.edu.au.
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Abstract |
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Key Words: malaria Plasmodium falciparum chloroquine drug resistance PfCRT drug/metabolite transporter
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Introduction |
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Two proteinsPgh1 (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 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.
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Materials and Methods |
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Alignment of Hydropathy Profiles
Hydropathy profile alignments were generated at http://bioinformatics.weizmann.ac.il/hydroph/ using Kyte-Doolittle (x 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 Adobe® Photoshop® 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 membrane-spanning 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 MacVectorTM 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.
Phylogenetic Analyses
Regions of the alignment that could not be aligned unambiguously were excluded prior to analysis. A phylogenetic tree was estimated using the Neighbor-Joining method (Saitou and Nei 1987) and uncorrected ("p") amino acid distances in MacVectorTM 7.1. Ties in the tree were resolved randomly and a bootstrap analysis (Felsenstein 1985) was performed with 1,000 replicates.
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Results and Discussion |
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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, 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.
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The best tree was estimated using the Neighbor-Joining 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 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.
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For the RhaT, GRP, and CEO proteins it is the odd-numbered 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]).
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 Gerardy-Schahn 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 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).
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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 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 intra-TMD 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 UDP-galactose 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.
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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 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 H+-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 H+-coupled), but this remains to be tested.
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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 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 CQ-sensitive 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 H+ 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.
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Acknowledgements |
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Footnotes |
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References |
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