CHAPTER 3 Bioinformatic studies and antigenic peptide selection for the putative
3.2 Results
3.2.4 Identification of a putative Plasmodium spp. Cox17 copper metallochaperone
The copper metallochaperone Cox17 has been implicated in the delivery of copper to the mitochondrion in yeast and mammalian cells (Cobine et al., 2006a; Leary et al., 2009).
Characterisation of the Cox17 metallochaperone has made use of the S. cerevisiae model, although mammalian isoforms have also been characterised. To date, a Cox17 chaperone has not yet been annotated or characterised in any Apicomplexan organism. To identify a Plasmodium Cox17 ortholog, the Arabidopsis thaliana Cox17 sequence was used to BLASTp search PlasmoDB. This particular sequence was selected since the A. thaliana genome appears closely related to the Plasmodium genome (Gardner et al., 2002) This search revealed a putative Cox17 sequence in the genomes of P. falciparum, vivax, yoelii, berghei, chabaudi, galinaceum, reichenowi and knowlesi. These were the same species predicted to encode a copper transporter. A ClustalWTM alignment of all retrieved parasite Cox17 sequences, with the sequences of known Cox17 proteins (Figure 3.4), was constructed to identify conserved amino acids.
Hs MPGLVDS---NPAPPE---SQEKKPLK-P CC A C PETKKARDACIIEKGE--EHCGHLIEAHKECMRALGFKI---- 63 Sc MTETDKK---QEQEN---HAECEDKPKP CC V C KPEKEERDTCILFNGQDSEKCKEFIEKYKECMKGYGFEVPSAN 69 At MTDQPAQN-GLIPPPTSEPSKAAASAETKPKKRICCACPDTKKLRDECIVEHGE--SACTKWIEAHKICLRAEGFNV---- 74 Pf MGMSLNK---PINNTN---EANKGEVKKKKICCVCLETKKLRDECIVKLGE--EQCKKFIDDHNKCLRSEGFDIK--- 67 Py MGLGLTK---PLNTT----EESKTCAKKKKICCVCLDTKKLRDECIVNLGE--EQCKKYINDHNQCLRNEGFDIK--- 66 Pv MGFF-NW---PSKNTS---EEAKGGAAKKKICCVCLETKKLRDECIVKLGE--EQCRKYIEDHNQCLRDEGFDVK--- 66 Pb MGLSLTK---PLNTT----EENKTCAKKKKICCVCLDTKKLRDECIVNLGE--EKCKKYIDEHNQCLRNEGFDIK--- 66 Pk MGFFSNW---PFKNTS---EEAKGGAPKKKICCVCLETKKLRDECIVKLGE--EQCRKYIEDHNQCLRNEGFDVK--- 67 Pc MGSSLT---ILNTT----DESKTGAPKKKICCVCLDTKKLRDECIVKYGE--KKCKKYIDDHNRCLRNEGFDIK--- 65 Pg MGTFLNK---PIKNTN---EEIKGE-KKKKICCVCLETKKLRDECIVKLGE--EQCKKFIDDHNQCLRNEGFDIK--- 66 Pr MGMSLNK---PINNPN---EANKGEVKKKKICCVCLKTKKLKDECIVKLGE--KQCKKFIDDHNKCLRSEGFDIK--- 67 * **.* *: :* ** *: . * . *: :: *:: ** :
Figure 3.4 Alignment of conserved amino acids in human, yeast and Arabidopsis Cox17 sequences with the Plasmodium sequences
Cox17 sequences aligned are for Homo sapiens (Hs), Saccharomyces cerevisiae (Sc), Arabidopsis thaliana (At), P.
falciparum (Pf), P. yoelii (Py), P. vivax (Pv), P. berghei (Pb), P. knowlesi (Pk), P. chabaudi (Pc), P. galinaceum (Pg) and P. reichenowi (Pr) (Refer to Section 2.3 for the sequence accession numbers). Essential residues are highlighted by colour. Conserved residues are indicated by *, whilst : represents a conserved substitution and . a semi-conserved substitution. The underlined sequence on the Hs and Sc sequences represents the Cox17 copper binding site.
A mutagenic study on more than two-thirds of the S. cerevisiae Cox17 protein sequence identified that only one-sixth of these mutants produced a respiration-deficient phenotype (Punter and Glerum, 2003). Surprisingly, only 40% of the conserved residue mutations had an adverse effect on Cox17 function. The conserved residues that were affected by mutation are Cys-23, Cys-24, Cys-26, Arg-33, Asp-34, Cys-47 and Cys-57 (Punter and Glerum, 2003).
Alignment of the eight retrieved Plasmodium spp. sequences with the human and yeast Cox17 sequences, established that these essential residues were conserved (Figure 3.4). The one exception was for the P. reichenowi sequence where a conserved substitution replaced Arg-33 for a Lys residue. Considering sequencing of the P. reichenowi genome is incomplete this difference could be the result of a sequencing error or artefact. It is important to note the conservation of the CysCysXaaCys motif at amino acids 23 to 26 (S. cerevisiae numbering) in all the Plasmodium sequences. This motif forms part of the Cox17 copper binding domain (underlined sequence in Figure 3.4) and is essential to its function (Abajian et al., 2004; Heaton et al., 2000; Punter and Glerum, 2003).
Figure 3.5 P. falciparum Cox17 homology model
The Saccharomyces cerevisiae Cox17 structure (green) (Arnesano et al., 2005) is included for comparison (A). P.
falciparum Cox17 (brown) was modelled against the NMR-solved structure of Homo sapiens Cox17 (yellow) without (B) or with (C) copper bound (Banci et al., 2008). The model was constructed by sequence alignment (D). In panels A to C the amino (Nt) and carboxy (Ct) termini are labelled. Coloured residues, in all three panels, are shown as stick representations and correspond to the residues highlighted in D, although residue numbers differ in panel A as does an additional cysteine residue (coloured yellow). The underlined cysteine doublet in D is coloured white in all panels. Copper in panel C is shown by the red sphere (●).
To understand Plasmodium Cox17 function, the Homo sapiens Cox17 (hCox17) NMR structure (Banci et al., 2008b) was used as a template to model the hypothetical structure of P.
falciparum Cox17 (PfCox17) (Figure 3.5) with the Swiss-pdb DeepView program (Guex and Peitsch, 1997). Both yeast and human Cox17 have a coiled coil-helix-coiled coil helix structural motif (Figure 3.5) (Abajian et al., 2004; Arnesano et al., 2005; Banci et al., 2008b), which is predicted to be common to the related mitochondrial proteins Cox19 (Nobrega et al., 2002) and Cox 23 (Barros et al., 2004). From the homology model of PfCox17 this structural motif appears to be conserved, with a close resemblance observed between PfCox17 and both the apo and Cu-loaded forms of hCox17 (Figure 3.5b and c, respectively). Located at the amino and carboxyl terminal ends of each helix are four conserved cysteine residues that form two interhelical disulfide bonds in yeast and human Cox17 (Figure 3.5, red and cyan highlighted residues). These conserved residues are also present in PfCox17 (Figure 3.5d) and appear to map closely to the corresponding residues in both apo and Cu-loaded hCox17 (Figure 3.5b and c, respectively). The conserved and essential Arg-32 and Asp-34 residues in hCox17 are also
HsCox17 MPGLVDS---NPAPPESQEKKPLKPCCACPETKKARDACIIEKGEEHCGHLIEAHKECMR 57 PfCox17 MGMSLNKPINNTNEANKGEVKKKKICCVCLETKKLRDECIVKLGEEQCKKFIDDHNKCLR 60 * ::. *. .:. * * * **.* **** ** **:: ***:* ::*: *::*:*
HsCox17 ALGFKI- 63 PfCox17 SEGFDIK 67 : **.*
Nt
Ct
Ct
Nt
Nt
Ct
B.
A. C.
D.
closely mapped by the corresponding residues in PfCox17 (Figure 3.5b and c). Copper binding to hCox17 results in local structural rearrangements around the two copper-coordinating cysteine residues (Figure 3.5b and c, white highlighted residues) causing the normally unstructured and flexible amino terminus (Figure 3.5b) to become more structured and less flexible (Figure 3.5c) (Banci et al., 2008b). PfCox17 appears to closely resemble these changes in protein structure. The one clear difference between PfCox17 and hCox17 is the insertion of three amino acids in the unstructured amino terminus (Figure 3.5b and d). However, this appears to have little bearing on the structure predicted for PfCox17.
3.2.5 Analysis of the putative P. falciparum Cox17 metallochaperone coding sequence