The plant-type
ferredoxin/
ferredoxin-NADP(+) reductase (Fd/FNR) redox system found in parasites of the phylum Apicomplexa has been proposed as a target for novel drugs used against life-threatening diseases such as
malaria and
toxoplasmosis. Like many
proteins from these protists, apicomplexan FNRs are characterized by the presence of unique
peptide insertions of variable length and yet unknown function. Since three-dimensional data are not available for any of the parasite FNRs, we used limited proteolysis to carry out an extensive study of the conformation of Toxoplasma gondii FNR. This led to identification of 11
peptide bonds susceptible to the action of four different
proteases. Cleavage sites are clustered in four regions of the
enzyme, which include two of its three species-specific insertions. Such regions are thus predicted to form flexible surface loops. The
protein substrate Fd protected FNR against cleavage both at its N-terminal
peptide and at its largest sequence insertion (28 residues). Deletion by
protein engineering of the species-specific subdomain containing the latter insertion resulted in an
enzyme form that, although catalytically active, displayed a 10-fold decreased affinity for Fd. In contrast, removal of the first 15 residues of the
enzyme unexpectedly enhanced its interaction with Fd. Thus, two flexible
polypeptide regions of T. gondii FNR are involved in Fd interaction but have opposite roles in modulating the binding affinity for the
protein ligand. In this respect, T. gondii FNR differs from plant FNRs, where the N-terminal
peptide contributes to the stabilization of their complex with Fd.