Recombinant expression and initial characterisation of two Plasmodium copper binding proteins.
Plasmodium falciparum is a protozoan parasite responsible for the most severe form of human malaria, with infection often resulting in death. Efforts to control malaria have been hindered by an increased spread of parasite resistance to previously effective antimalarial drugs, leading to an intensified search for novel antimalarial drug targets. A group of proteins suggested as potentially effective targets are the integral membrane transport proteins, since they play key roles in Plasmodium parasite growth and replication. One such membrane protein recently characterised was the P. falciparum copper efflux transporter. Treatment of cultured P. falciparum parasites with the intracellular copper chelator neocuproine inhibited parasite growth, suggesting that additional mechanisms for malaria parasite copper homoeostasis are likely to be present. Copper is an essential trace element involved in enzymatic processes requiring redox-chemistry. In higher eukaryotes copper is transported across the plasma membrane via the copper transport protein, Ctr1, and distributed intracellularly by copper metallochaperones. The mechanisms for copper acquisition and distribution in the Plasmodium parasite are, however, yet to be characterised. An in silico Basic Local Alignment Search Tool for protein (BLASTp) screen of the Plasmodium database (www.plasmodb.org) identified sequences corresponding to a putative copper transporter, and associated copper metallochaperones, in eight species of the Plasmodium parasite. Each of the Plasmodium copper transport protein sequences was found to contain features common to the well characterised copper transporters. These features included predicted copper-binding motifs in the protein's amino terminus, three membrane spanning domains and the characteristic MxxxM and GxxxG motifs located in the second and third transmembrane domains, respectively. Affinity purified anti-peptide antibodies, generated against an immunogenic peptide (CSDKQSGDDECKPILD) in the amino terminus of a putative malaria parasite copper transporter (PY00413), detected the target protein in murine malaria parasites in association with a parasite membrane. The open reading frames corresponding to the amino terminal domains of one P. berghei [PBANKA_130290 (447 bp)] and two P. falciparum [PF14_0211 (132 bp) and PF14_0369 (282 bp)] putative copper transport proteins were PCR amplified, ligated into pGEM®-T and then expressed as recombinant fusion proteins with maltose binding protein (MBP). The resulting sizes for the recombinant proteins were 61kDa for MBP-PbCtrNt, 48kDa for MBP-PfCtr211Ntᵀᴰ and 55kDa for MBP-PfCtr369Ntᵀᴰ, with each protein being recognised by a corresponding anti-peptide antibody. All three recombinant proteins bound copper in vitro and in vivo, with each having a binding preference for the reduced cuprous ion. This preference has been similarly established for the characterised copper transporters. Although the results supported the expression and copper binding ability of a Plasmodium parasite copper transport protein, the directional transport of copper, by this protein, requires experimental confirmation as does its specific location. The identification of a P. falciparum copper transporter, and other copper dependent proteins, implies a parasite metabolic requirement for copper. Mammalian and yeast cells require a Cox17 metallochaperone for copper delivery to cytochrome-c oxidase. Identification of P. falciparum orthologs for Cox17 (PF10_0252) and a number of cytochrome-c oxidase subunits (PF13_0327; PF14_0288; mal_mito_1; mal_mito_2; PFI1365w; PFI1375w), suggests the existence of similar parasite mechanisms for copper delivery. Analysis of the Plasmodium Cox17-like sequences identified essential amino acids conserved in the well characterised yeast and mammalian Cox17. This included the identification of six cysteine residues essential for Cox17 function. A homology model of P. falciparum Cox17, with human Cox17 as the template [PDB ID: 2RN9 (apoCox17); 2RN8 (Cu⁺-Cox17)], suggested that Plasmodium Cox17 orthologs would adopt a similar structural conformation. The open reading frames for full-length P. yoelii [PY03823 (192 bp)] and P. falciparum [PF10_0252 (195 bp)] Cox17 were PCR amplified, ligated into pGEM®-T and then expressed as recombinant fusion proteins with either a His₆-tag or glutathione S-transferase (GST)-tag, respectively. The resulting sizes for the recombinant proteins were 11.6kDa for His₆-PyCox17 and 33.5kDa for GST-PfCox17, with each protein being recognised by a corresponding anti-peptide antibody. Both recombinant Cox17 proteins bound the cuprous ion in vitro and in vivo, similar to mammalian and yeast Cox17. This supported the likely existence of a mitochondrial copper metallochaperone pathway within the malaria parasite; however, this requires further experimental confirmation. Identification of a parasite copper transport protein, and associated metallochaperones, could provide novel targets for drug-based inhibition of parasite growth. Alternatively, the copper transporter may provide a novel mechanism for drug delivery into the Plasmodium parasite. The potential of these malaria parasite proteins being effective drug targets does, however, remain to be confirmed.