Deciphering the atomic structure and functions of HCF101 proteins through an integrative biological approach targeting two evolutionarily-divergent orthologs – HCFforLIFE

Iron-sulfur (Fe-S) clusters are universal co-factors embedded in proteins belonging to different families, most of them supporting fundamental biological functions. Efficient Fe-S delivery to proper client apoproteins is ensured by complex cellular machineries involving few dozens of late-acting Fe-S carrier proteins that supply hundred of client apoproteins with Fe-S clusters. Our transdisciplinary HCFforLIFE project aims at deciphering the functions of such essential but poorly characterized Fe-S cluster carrier proteins belonging to the NTPase superfamily, the so-called HCF101 proteins, selected from two model eukaryotes with different evolutionary origin and life style: the plant Arabidopsis thaliana (At) and the apicomplexan parasite Toxoplasma gondii (Tg).

HCF101 (High Chlorophyll Fluorescence 101) proteins are restricted to algae, terrestrial plants, and apicomplexan parasites. First discovered in Arabidopsis, the plastidial AtHCF101 was inferred to Fe-S cluster delivery from corresponding Athcf101 knockout lethal mutants which are devoid of the sole [4Fe-4S] subunits of the photosystem I (PSI) and therefore unable to perform photosynthesis. HCF101 is also essential for fitness of the human parasite T. gondii. And yet, in spite of their crucial role in sustaining viability, HCF101 structural features, exact functions, mode of action and client proteins still remain to be characterized.

The sparse information available on HCF101 makes these proteins enigmatic in many ways. Apicomplexa possess an ancestral plastid, called the apicoplast whose photosynthetic function has been lost during evolution. Intriguingly, our preliminary results and predictions indicate that TgHCF101 resides in the cytosol, which strongly questions the role of HCF101 in this compartment. All HCF101 proteins exhibit a typical organization with three domains. However, N-terminal and C-terminal regions have unknown function, while the central domain affiliates HCF101 to a class of P-Loop NTPases, an activity still to be proven.

We hypothesize that At and Tg HCF101 share the organizational conservation of their domains, and a common 3D fold related to a conservation of their biochemical properties and NTPase-dependent Fe-S clusters delivery. Yet, because they reside in different compartments, they are likely involved in Fe-S transfer to a different repertoire of client proteins, a specificity likely driven by particular changes in the HCF101/partner interaction interface.

To fulfill the important gap of knowledge on these essential proteins, we propose to:
• Determine At and Tg HCF101 protein structures,
• Establish the proteomes of hcf101 mutants and the At and Tg HCF101 interactomes to reveal Fe-S client proteins,
• Establish the structure of selected HCF101/interactor complexes, and identify structural determinants using site-directed mutagenesis and transgenesis,
• Analyzing HCF101 redox properties and assess their NPTase activity.

Our study of HCF101 from phylogenetically distant organisms offers a unique opportunity to decipher the role and client proteins of this essential component of Fe-S assembly machineries with new molecular and structural information. Our project brings experts in protein structures, plant molecular biology & biochemistry, and human parasitology with robust approaches and cutting-edge technologies. Given the importance of Fe-S cofactors for photosynthesis and other key metabolic pathways, our results will open huge perspectives to promote plant growth as HCF101 is likely crucial for crop productivity and health. HCF101 proteins do not exist in mammals, making TgHCF101 protein a good candidate as a potential drug target for Toxoplasm control. Thus, our project also offers an opportunity to enrich the therapeutic arsenal against parasites that infects ~one third of the world’s human population and many vertebrates, causing serious and even fatal complications such as abortion and encephalitis.