Toxoplasma gondii: analysis of the active site insertion of its ferredoxin-NADP(+)-reductase by peptide-specific antibodies and homology-based modeling.
Apicomplexan parasites possess an apicoplast-localized redox system consisting of a plant-type ferredoxin-NADP(+)-reductase (FNR) and its redox partner ferredoxin, a small [2Fe-2S] protein. We show here that several apicomplexan FNRs contain unique amino acid insertions of various lengths which are located in close proximity to the enzymatically important FAD and ferredoxin-binding sites of these proteins. Using the insertion of the Toxoplasma gondii reductase as an example we raised epitope-specific antibodies against an 11 amino acids long peptide predicted to be surface-exposed within this insertion. This peptide was found to be immunogenic when presented to the immune system as part of a carrier protein, but also in its natural structural context in the whole recombinant protein, implying that the epitope is surface-exposed. Three-dimensional modeling of T. gondii FNR based on the known 3D-structure of maize root FNR predicts that the overall structure of plant and apicomplexan FNRs are very similar and that the 11 amino acids are part of an alpha-helix, looping out of the molecule. Collectively, these data suggest that the insertion in T. gondii FNR does not affect the overall structure of the protein but may have an effect on the binding dynamics of FAD, NADP(+), and/or ferredoxin to FNR.
Bednarek
Anika
A
FB Biologie/Parasitologie, Philipps-Universität Marburg, Karl-von-Frisch-Str., D-35032, Marburg, Germany.
Wiek
Sabine
S
Lingelbach
Klaus
K
Seeber
Frank
F
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005260:Female; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007106:Immune Sera / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017510:Protein Folding; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D014122:Toxoplasma / Q000201:enzymology*
D000939:Epitopes; D007106:Immune Sera; D011994:Recombinant Proteins; D005287:Ferredoxin-NADP Reductase