Structural Elucidation of the Interaction of Apicoplast Resident Ferredoxin and its Interacting Proteins

Abstract

The apicoplast is an essential plastid-like organelle which was derived from red algae by secondary endosymbiosis and found in members of the Apicomplexa phylum (except Cryptosporidium and most gregarines). This organelle harbours several essential metabolic pathways absent from the parasite’s host making it and the pathways therein prime targets for the design of drugs against diseases such as malaria (caused by Plasmodium spp.) and toxoplasmosis (caused by Toxoplasma gondii). Inside the apicoplast, an electron transfer system is essential for various enzymatic processes. This system is facilitated by the ferredoxin redox system, which comprises the plant- type ferredoxin-NADP⁺ reductase (ptFNR) and its redox partner, plant-type ferredoxin (ptFd). Whereas protein-protein interaction is known to mediate the electron transfer from ptFd to partner enzymes, the exact amino acid residues at the interaction interface remains unknown. In this study, the exact amino acid residues at the interaction interface between ptFd and one of its partner enzymes in the methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis called (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase (Particularly PfIspH) was determined using the combination of an E. coli model and an advance computational biology technique that helped navigate the challenge of performing protein-protein interaction with labile Fe-S containing proteins and structural determination of protein complexes respectively. The results reaffirmed the role of electrostatic interaction in the protein-protein interaction and provides vital information for the design of inhibitors against such protein complexes. The result of the E. coli model screen was recapitulated in the apicoplast of T. gondii making a case for the model as a very useful first screening platform for functional mutations or drug effects before more laborious and time-consuming assays in the parasite are performed. Further application of the model in flavodoxin complementation study provides evidence for the evolutionary relationship between Cyanobacteria (Nostoc), Chromera velia and Apicomplexa and how the parasitic lifestyle of apicomplexan parasites may likely influence the flexibility of their redox system. Furthermore, report on the inability of flavodoxins to complement TgFd provide indirect evidence for the involvement of ptFd in the SUF (sulfur utilization factor) pathway of Fe- S biogenesis in the apicoplast. Hence, the findings of this study contribute to a better understanding of the interaction interface between PfFd and PfIspH, the evolutionary relationship between flavodoxins and ptFd as well as the metabolic role of ptFd as an essential, central electron distributing hub in T. gondii and P. falciparum, supporting its importance for the parasite’s metabolism and underlining its potential as a drug target in apicomplexan parasites.