Biophysical analysis ofPlasmodium falciparumHsp70-Hsp90 organizing protein (PfHop) reveals a monomer that is characterised by folded segments connected by flexible linkers

Abstract

Plasmodium falciparum causes the most lethal form of malaria. The cooperation of heat shock protein (Hsp) 70 and 90 is important for folding of a select number of cellular proteins that are crucial for cyto-protection and development of the parasites. Hsp70 and Hsp90 are brought into a functional complex that allows substrate exchange by stress inducible protein 1 (STI1), also known as Hsp70-Hsp90 organizing protein (Hop). P. falciparum Hop (PfHop) co-localises and occurs in complex with the parasite cytosolic chaperones, PfHsp70-1 and PfHsp90. Here, we characterised 2 the structure of recombinant PfHop using synchrotron radiation circular dichroism (SRCD) and small-angle X-ray scattering. Structurally, PfHop is a monomeric, elongated but folded protein, in agreement with its predicted TPR domain structure. Using SRCD, we established that PfHop is unstable at temperatures higher than 40 °C. This suggests that PfHop is less stable at elevated temperatures compared to its functional partner, PfHsp70-1, that is reportedly stable at temperatures as high as 80 °C. These findings contribute towards our understanding of the role of the Hop-mediated functional partnership between Hsp70 and Hsp90.

Inhibition of Plasmodium falciparum Hsp70-Hop partnership by 2-phenylthynesulfonamide

“…We previously resolved the structure of PfHop using SAXS analysis. Our previous study demonstrated that the TPR domains of PfHop assemble like "beads on a string'' (Makumire et al, 2020). This arrangement allows Hsp70 and Hsp90 to slide through the concave-shaped TPR domains to facilitate interaction.…”

“…An additional construct, pQE30-PfHsp70-1 NBD encoding for the NBD of PfHsp70-1 was similarly used to express the truncated version of this chaperone (Zininga et al, 2016). The production of recombinant protein was induced by isopropylthiogalactoside (IPTG) and the protein was purified using sepharose nickel affinity chromatography (Makumire et al, 2020(Makumire et al, , 2021. The expression and purification of recombinant proteins were analyzed by SDS-PAGE and confirmed by Western blot analysis using α-PfHsp70-1 (Shonhai et al, 2008), α-PfHop (Gitau et al, 2012), and α-His antibodies, respectively.…”