CHAPTER 5 The Plasmodium spp. putative Cox17 copper metallochaperone
5.3 Discussion
5.3.3 Copper binding to His 6 -PyCox17 and GST-PfCox17
Mammalian Cox17, which contains six conserved cysteine residues, can in principle exist in three different oxidation states; the fully oxidised protein with three disulfide bonds (Cox173S-S), a partially oxidised form with two disulfide bonds (Cox172S-S) or a fully reduced state where no disulfide bonds (Cox170S-S) are present (Palumaa et al., 2004; Voronova et al., 2007a). These various forms of Cox17 have been found to differ in their copper binding ability. The partially oxidised state binds a single Cu+ ion, whereas the fully oxidised state cannot bind copper (Palumaa et al., 2004). On the other hand, the fully reduced form of Cox17 contains six free cysteine residues that cooperatively bind four Cu+ ions to form a tetracopper-thiolate cluster (Arnesano et al., 2005; Palumaa et al., 2004; Voronova et al., 2007b). Recombinant bacterial expression of human Cox17, in an unaltered cellular redox environment, was found to yield three almost equally populated redox states corresponding to Cox172S-S, Cox171S-S and Cox170S-S
(Voronova et al., 2007b). Based on copper transfer studies, the preferred cellular redox state of Cox17 appears to be the partially oxidised state, Cox172S-S (Banci et al., 2008a). This state can been attained, in vitro, by treating purified Cox17 with millimolar concentrations of DTT since Cox17 contains two stable disulfide bonds and one labile bond that is susceptible to reducing agents (Arnesano et al., 2005; Banci et al., 2008b; Voronova et al., 2007a). Purified His6- PyCox17 and GST-PfCox17 were therefore treated similarly to promote this redox state (Cox172S-S) for in vitro copper binding studies.
Analysis of in vitro copper binding to His6-PyCox17 and GST-PfCox17 revealed both proteins could bind copper following protein purification (Figure 5.9a). The differences in copper binding between the His6-tagged and GST-fusion proteins has also been reported for yeast Cox17
(Heaton et al., 2001; Srinivasan et al., 1998). GST-tagged yeast Cox17 bound two molar equivalents of Cu+ (Srinivasan et al., 1998) whilst untagged Cox17 bound three molar equivalents (Heaton et al., 2001). This was suggested to be a result of the obligate dimeric state of GST (Heaton et al., 2001). An important difference between the present study, and those with yeast Cox17, is that copper binding was examined in vivo for yeast Cox17.
Therefore another explanation for the differences (Figure 5.9a) is that a mixed distribution of protein redox states was created, following DTT treatment, thereby causing differences in copper binding. Alternatively, DTT itself may have leached bound copper from the recombinant protein, similar to what was observed for Cu4Cox17 when DTT was used at supramillimolar concentrations (Palumaa et al., 2004). A further possibility is that these differences relate to truncated GST-PfCox17 accounting for part of the 10 µM protein solution. This could have reduced the amount of protein able to bind copper in solution.
His6-PyCox17 and GST-PfCox17 were also found to bind copper in a cellular environment (Figure 5.9b). A comparison of the in vivo data with the in vitro reconstitution of each protein indicated a similar difference between proteins with regard to the amount of copper bound.
GST dimerisation may have contributed to these differences, as reported for yeast Cox17, since it can result in a non-native conformer of Cox17 being induced by Cu+ binding (Heaton et al., 2001). It is also possible that copper ions bound in vivo were lost during purification and dialysis, as reported for native yeast Cox17 purification (Beers et al., 1997). Through a slight modification of the BCA assay it was shown that both recombinant proteins bound the cuprous ion under the two conditions examined. This finding is in agreement with the preferred redox state of copper bound to mammalian and yeast Cox17 (Abajian et al., 2004; Banci et al., 2008b; Beers et al., 1997; Palumaa et al., 2004).
The ascorbic acid oxidation assay has previously been used to demonstrate the copper binding ability of a methionine peptide (Jiang et al., 2005) and was used to demonstrate copper binding to the amino terminal domains of the P. berghei and P. falciparum copper transporters (Section 4.2.4). Ascorbic acid is stable to aerial oxidation, but the presence of metal ions, such as iron or copper, catalyse its oxidative degradation (Jiang et al., 2005). The oxidation rate of ascorbic acid can be monitored between 245 and 265 nm (Buettner, 1988). Conveniently the oxidation rate of ascorbate is known to decrease in the presence of metal chelators with slower oxidation rates indicative of more stable chelates (Khan and Martell, 1967). Metal chelation by the Plasmodium copper transporters was thought to be via methionine motifs present in the protein sequence, but this was not confirmed. Plasmodium Cox17 recombinant proteins are predicted to coordinate copper via a similar cysteine motif to that found in yeast Cox17 (Abajian et al., 2004).
The ability of His6-PyCox17 and GST-PfCox17 to inhibit copper-catalysed ascorbic acid oxidation was tested in vitro (Figure 5.10). Both proteins inhibited ascorbate oxidation and showed a near identical decrease in the oxidation rate. The reduction in the oxidation rate suggests stable chelation of what is presumed to be the cuprous ion. Although the ascorbate UV-vis assay alone does not prove the preferred copper oxidation state for these Plasmodium proteins, when analysed in conjunction with the previously described in vivo assay it suggests the cuprous ion is the preferred substrate. Given the reducing potential of both the prokaryotic and eukaryotic cell cytoplasm (Schafer and Buettner, 2001) it is predicted that the Cu+ ion is the likely oxidation state in which copper would be found within the cell (Davis and O'Halloran, 2008). A similar reducing cytoplasmic environment has been identified for the Plasmodium parasite (Krnajski et al., 2001). If copper is present in a reduced state within the parasite cytoplasm, it is the likely substrate for cytoplasmic copper-requiring proteins. Parasite Cox17 is presumed to be located in the cytoplasm based on previous findings (Beers et al., 1997).
In the present study P. yoelii and P. falciparum Cox17 were recombinantly expressed and shown to bind copper, but further protein characterisation is necessary. Firstly, native parasite Cox17 requires localisation using an antibody-mediated approach. This can be achieved using confocal and/or transmission electron microscopy. Alternatively, Cox17 localisation could be determined by attaching it to a fluorescent GFP-tag. Secondly, to assist with establishing protein function, Plasmodium Cox17 could be used in a yeast functional complementation system using cox17Δ cells. Thirdly, site-directed mutatgenesis could be employed to help identify the amino acid residues important for Cox17 structure and function. A final possibility would be to attempt producing protein crystals of Plasmodium Cox17, with and without bound copper, to help establish the protein's copper binding properties in more detail.
CHAPTER 6