Characterization of the structure-function features of the R2TP complex of Plasmodium falciparum

Abstract

Malaria remains a major cause of death worldwide. Plasmodium falciparum is responsible for the most severe form of malaria. The malaria parasite life cycle involves stages of development that span across the cold-blooded mosquito vector and a warm-blooded human host. Maintaining protein homeostasis across these physiologically distinct life stages of the parasite is mainly accomplished by a family of proteins termed molecular chaperones that assist in protein folding. Heat shock protein 90 (Hsp90) a molecular chaperone of P. falciparum is known to be important for cell development and signal transduction. The protein clientele of this chaperone runs in several hundred and efforts are ongoing to account for all its cellular clients. R2TP complex acts as a chaperone or the assembly of critical complexes in the cell, hence it is implicated in the assembly of the Hsp90 functional complex. Hence, this study aims to characterize the structural-functional features of the R2TP complex of P. falciparum. Bioinformatics analysis has revealed unique features of this complex with 3 RUVBL genes as opposed to its orthologues from yeast and humans whom both have 2 Rvb and 2 RUVBL genes, respectively. The study involved the design of antibodies and biophysical characterization of PfRUVBL proteins and their functional features. Biophysical characterization of PfRUVBL1, PfRUVBL2, and yeast Rvb1 was conducted using tryptophan spectrophotometry and limited proteolysis. In addition, surface plasmon resonance (SPR) was used to explore the oligomerization of the proteins. The ATPase activities of PfRUVBL1 and PfRUVBL2 were further investigated. Furthermore, the holdase chaperone activities of the proteins were investigated by monitoring the capability of the proteins to suppress heat-induced aggregation of a model protein, malate dehydrogenase (MDH). P. falciparum recombinant proteins showed abilities to bind nucleotides and revealed ATPase activity. In addition, these proteins revealed capabilities to self and hetero associates as suggested by SPR and slot blot assays.