CHAPTER 4 The Plasmodium spp. putative copper transport protein: Native protein
4.3 Discussion
4.3.5 Copper binding to MBP-PbCtrNt, MBP-PfCtr211Nt TD and MBP-PfCtr369Nt TD
Similar to MBP-PbCtrNt, copper binding studies using MBP-PfCtr211NtTD and MBP- PfCtr369NtTD indicated that both proteins bound copper in vitro and in vivo (Figure 4.21) with an apparent preference for the cuprous ion. Copper is presumed to be coordinated to methionine motifs (MxM or MxxM) present in each of these Plasmodium proteins. These motifs have been demonstrated to be essential for the function of the yeast and human copper transporters when extracellular copper is limited (Eisses and Kaplan, 2005; Guo et al., 2004; Puig et al., 2002a).
The amino terminus of the yeast copper transporter contains eight such methionine motifs, but only the last methionine of the eighth motif is essential for function (Puig et al., 2002a). This particular methionine has been found to be conserved in other characterised copper transporters and is located 20 amino acids from the first transmembrane domain. Analysis of the two P. falciparum copper transporters identified one MxxM motif in PfCtr211NtTD and one MxM motif in PfCtr369NtTD (Figure 4.13). Based on hydropathy plots (Figure 3.3) it was established that the last methionine of each of these motifs was located 20 amino acids from the first predicted transmembrane domain. This suggests that this motif may be involved in copper coordination. However, the involvement of other residues in copper coordination cannot be excluded. This is of particular importance since a number of cysteine and histidine residues are also present in the amino-terminal sequences of the Plasmodium copper transporters. Both cysteine and histidine residues have been implicated in metal ion coordination in other proteins (Davis and O'Halloran, 2008; Eisses and Kaplan, 2005).
In contrast to the P. falciparum proteins, the amino terminal domain of the P. berghei copper transporter contains two MxM motifs. The second of these two motifs contains a methionine residue 20 amino acids from the first predicted transmembrane domain, therefore implicating this motif in copper coordination. However, copper coordination to the other methionine motif, or to histidine and cysteine residues, cannot be excluded. Preliminary electron spin resonance data, from MBP-PbCtrNt copper binding studies, suggested that copper was coordinated by
two histidines, a cysteine and methionine residue (data not shown). Identification of the specific residues involved in copper coordination to the Plasmodium proteins requires the use of more sophisticated techniques such as protein crystallisation or nuclear magnetic resonance (NMR) spectroscopy. These techniques could also be used to confirm the folded state of each recombinant protein, which would help determine the importance of correct protein folding to copper coordination.
When expressed in the presence of copper, the final yield of MBP-PfCtr211NtTD and MBP- PfCtr369NtTD was significantly increased (Figure 4.22). A similar phenomenon was, however, not observed when MBP-PbCtrNt was expressed in the presence of copper. This observation could perhaps be accounted for by the fact that MBP-PbCtrNt appears to aggregate in the E.
coli host cell. However, the fact that MBP-PbCtrNt was still capable of binding copper in vivo (Figure 4.20b), despite its aggregation, suggests other factors influenced these differences.
One likelihood is the differing copper requirements of the cellular compartments to which the respective proteins were targeted, namely the cytoplasm and periplasm. Almost all bacterial copper proteins, such as multi-copper oxidases, amine oxidases and lysine oxidases are found in the bacterial periplasm or excreted extracellularly (Rensing and Grass, 2003). It has even been proposed that E. coli cells do not require copper for cytoplasmic enzymes and only require copper for periplasmic or inner membrane enzymes, such as Cu,Zn-superoxide dismutase or cytochrome-c oxidase (Bagai et al., 2008). Since copper is required in the periplasm, efficient regulatory mechanisms are essential (Macomber et al., 2007). This is perhaps best highlighted by studies of two periplasmic proteins thought to be involved in copper homoeostasis, namely PcoA and PcoC. Mutation of either protein interferes with copper resistance (Brown et al., 1995) whilst gene deletion causes copper hypersensitivity (Huffman et al., 2002).
Due to the oxidising environment of the bacterial periplasm, tight control of copper ensures this metal ion cannot freely enter redox chemistry to produce harmful reactive oxygen species (Puig et al., 2002b). Under standard laboratory growth conditions, this implies there would be limited copper available in the periplasm for copper binding proteins that are not required for E. coli metabolic function. Consequently, MBP-PfCtr211NtTD and MBP-PfCtr369NtTD expression is perhaps limited to ensure native E. coli copper-dependent proteins can function normally under these growth conditions. However, if excess extracellular copper is added to the growth medium, MBP-PfCtr211NtTD and MBP-PfCtr369NtTD protein yield is improved (Figure 4.22) by alleviating the copper-related metabolic stress from the E. coli cells. The lack of an increase in protein yield for cytoplasmically expressed MBP-PbCtrNt, could perhaps be related to the fact that few cytoplasmic E. coli proteins require copper as an essential cofactor (Macomber et al.,
2007). Thus the cytoplasmic expression of MBP-PbCtrNt would place less stress on E. coli cellular metabolism under standard growth conditions, resulting in similar yields in the presence or absence of extracellular copper.
In mammalian and yeast cells the favoured redox state of copper for transfer and transport, is the reduced cuprous ion (Cu+) (Hassett and Kosman, 1995; Puig et al., 2002a). This is thought to be due to the fact that Cu+ is more exchange labile than Cu2+ thereby facilitating its transfer between two binding sites (Jiang et al., 2005; Rees and Thiele, 2007). Considering the highly reactive nature of Cu+ in an oxidising environment, the challenge is to safely sequester these ions prior to transport across the lipid bilayer. The methionine motifs present in the extracellular amino terminus of the copper transporter family are thought to provide this desired coordination environment (Jiang et al., 2005; Puig et al., 2002a). Methionine only coordination of Cu+ has been established through a unique study using a synthesized methionine motif peptide (MTGMKGMS). Inhibition of copper-catalysed ascorbic acid oxidation, by this peptide, suggested it chelated Cu+ thereby preventing it from participating in the redox cycle (Figure 4.23). This data, combined with similar experiments using peptide variants lacking methionines as well as electrospray mass spectra, strongly supported methionine-specific coordination of Cu+ (Jiang et al., 2005). The affinity with which these motifs bound copper (~2.5 µM) was also found to be compatible with biological copper uptake, which is between 1 and 5 µM in a variety of organisms (Puig and Thiele, 2002). These methionine motifs were therefore suggested to be required by copper transport proteins for extracellular copper acquisition and transport across the membrane.
The two P. falciparum copper transporter amino termini are each predicted to contain a single methionine motif (Figure 4.13). This motif is presumed to be involved in copper binding, as suggested from the in vitro and in vivo studies. The ability of the recombinant P. falciparum amino termini to inhibit copper-catalysed ascorbic acid oxidation was examined and indicated that both MBP-PfCtr211NtTD and MBP-PfCtr369NtTD inhibited ascorbic acid oxidation (Figure 4.24). Considering the dynamics of the redox cycle (Figure 4.23), this result suggested that each protein coordinated the Cu+ ion. Therefore, the ability of these recombinant proteins to readily bind copper combined with protein localisation studies (Figure 4.8) supports a physiological role for the Plasmodium copper transporters (Chapter 3). Nevertheless, cation uptake and/or functional complementation studies will be necessary to confirm the directional transport of copper by these parasite proteins.