Malaria threatens millions of people in mainly developing countries and caused over 400 thousand deaths in 2020 alone. The parasite Plasmodium falciparum is the dominant cause of malaria cases in Africa. The emergence of strains resistant to currently available treatments also creates an urgent need to find new targets.
However, the AAA+ ATPase enzyme p97 (aka VCP or CDC48), plays an essential role in cellular protein-homeostasis in eukaryotes and archaebacteria. This multimeric machine acts by segregating and unfolding improperly folded proteins from larger complexes, driven by the hydrolysis of ATP. This is followed by the action of the proteasome in the ubiquitin-proteasome pathway that subsequently leads to the degradation of the target protein. It is also implicated in other cellular processes such as membrane fusion, DNA repair and autophagy.
Two homologues of p97 in P. falciparum have previously been identified in the parasite. One localises in the cytoplasm (PFp97-Cyt) and likely carries out diverse tasks previously observed and characterised in other species. The second homologue in the parasite’s apicoplast (PFp97-Api) has lesser known cellular functions. These homologues have low sequence homology to human p97. Therefore, structural characterisation can inform our strategies for designing effective and specific inhibitors against them to target malaria parasite cells.
Recombinant PFp97 proteins were expressed in E. coli and purified using immobilised metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC). This was followed by cryo-electron microscopy (cryo-EM) to arrive at 3D reconstructions of the two proteins. This has given us given us structural insights into the oligomerisation and nucleotide-dependence of PFp97 proteins. Biochemical assays were used to investigate ATPase activity and the effect of p97 inhibitors. Altogether, these studies have provided us with a better understanding of the molecular mechanism of these ubiquitous and essential proteins in P. falciparum parasites.