Structure-function studies of the major insertion of the apicomplexan ferredoxin NADP+ reductase, investigated by mutagenesis and limited proteolysis

Apicomplexan parasites are a large phylum of unicellular and obligate intracellular organisms of great medical and economic importance. They include the human pathogens Plasmodium spp., the causative agents of malaria, and Toxoplasma gondii, an opportunistic human parasite. It was shown that most members of this phylum harbor a plastid-like organelle, called the apicoplast, of vegetal origin. The plant-type ferredoxin/ferredoxin-NADP+ reductase (Fd/FNR) redox system found in this organelle has been proposed as a target for novel drugs (1, 2). Elucidation of the properties and functions of this redox system would be greatly facilitated by a more detailed structural knowledge of the two components. Like many proteins from these protists, apicomplexan FNRs are characterized by the presence of unique peptide insertions of variable length and yet unknown function. The major insertion of T. gondii FNR (TgFNR) (28 aa with respect to maize root FNR) is near the FAD isoalloxazine ring binding motif. Thus, it is expected that it could influence the protein-protein interaction of the redox couple. The aim of this study was therefore to investigate the structure and the involvement of this region of TgFNR in substrate binding by mutagenesis and limited proteolysis approaches. All four proteinases tested yielded a limited number of peptides. Most of the cleavage sites were located within the major insertion of TgFNR, indicating that this region is surface exposed and is highly flexible. Furthermore, protection of the insertion by TgFd against proteolytic cleavage indicates a role for this region in ferredoxin binding (3). Three different deleted forms of the parasite enzyme were produced, in which the insertion was fully removed (Del1), partially removed (Del2) or replaced with the homologous region of plant FNR (Del3). Compared to the wild type enzyme, Del2 and Del3 showed similar values for the kinetic parameters of the TgFd-dependent NADPH-cytochrome c reductase activity, while Km for ferredoxin of Del1 was highly increased. By affinity chromatography on immobilized TgFd, deleted TgFNRs were shown to bind more weakly TgFd than the wild-type enzyme. Del1 showed a remarkable loss of affinity for ferredoxin, probably because it lacks three positively charged residues which are present in Del2 and in Del3 mutants. The results reported here allow us to conclude that the insertion of TgFNR is solvent exposed and structurally flexible. It is suggested to give rise to a 38-residue intrinsically unstructured subdomain, protruding from the top of the FAD-binding beta-barrel. Such a species-specific subdomain does not have major functions in protein folding and stability. Rather, it participates in TgFd binding, significantly increasing the stability of the protein-protein complex and improving the catalytic efficiency of the enzyme in the electron transfer to TgFd. 1. M. Vollmer et al. (2001) J. Biol. Chem. 276(8): 5483-5490 2. V. Pandini et al. (2002) J Biol Chem. 277(50):48463-48471. 3. V. Pandini et al. (2006) Biochemistry 45: 3563-3571