Comparative Characterization of Plasmodium falciparum Hsp70-1 Relative to E. coli DnaK Reveals the Functional Specificity of the Parasite Chaperone

Abstract

Hsp70 is a conserved molecular chaperone. How Hsp70 exhibits specialized functions across species remains to be understood. Plasmodium falciparum Hsp70-1 (PfHsp70-1) and Escherichia coli DnaK are cytosol localized molecular chaperones that are important for the survival of these two organisms. In the current study, we investigated comparative structure-function features of PfHsp70-1 relative to DnaK and a chimeric protein, KPf, constituted by the ATPase domain of DnaK and the substrate binding domain (SBD) of PfHsp70-1. Recombinant forms of the three Hsp70s exhibited similar secondary and tertiary structural folds. However, compared to DnaK, both KPf and PfHsp70-1 were more stable to heat stress and exhibited higher basal ATPase activity. In addition, PfHsp70-1 preferentially bound to asparagine rich peptide substrates, as opposed to DnaK. Recombinant P. falciparum adenosylmethionine decarboxylase (PfAdoMetDC) co-expressed in E. coli with either KPf or PfHsp70-1 was produced as a fully folded product. Co-expression of PfAdoMetDC with heterologous DnaK in E. coli did not promote folding of the former. However, a combination of supplementary GroEL plus DnaK improved folding of PfAdoMetDC. These findings demonstrated that the SBD of PfHsp70-1 regulates several functional features of the protein and that this molecular chaperone is tailored to facilitate folding of plasmodial proteins.

Mutation of GGMP Repeat Segments of Plasmodium falciparum Hsp70-1 Compromises Chaperone Function and Hop Co-Chaperone Binding

“…We further conducted the same assays on the DnaK- G derivative relative to wild type DnaK. Overall, the GGMP mutants of PfHsp70-1 exhibited similar CD spectra to that of wild type PfHsp70-1 protein ( Figure 3 A) characterised with minima at 222 nm and 209 nm, and maxima at 194 consistent with the reported α-helical nature of the protein [ 13 ]. This suggests that the wild type as well as the two select GGMP derivatives of PfHsp70-1 proteins were properly folded.…”

“…We recently established that PfHsp70-1 and E. coli DnaK exhibit unique structure-function features [ 13 ]. Although the SBD is known to play a role in defining the functions of these two proteins, the role of the GGMP in this regard is unknown.…”

“…One of the most intriguing aspects of members of the Hsp70 family of chaperones is that despite their general sequence conservation, they exhibit functional specificity [ 6 , 13 ]. The identification of the unique structure-function features of Hsp70 is therefore a priority in this regard.…”

“…We previously demonstrated that PfHsp70-1 possesses chaperone activity and that it physically associates with P. falciparum Hsp90 via Hop (PfHop) as a module [ 17 , 30 ]. In addition, we recently established that PfHsp70-1 exhibits unique structure-function features compared to DnaK [ 13 ]. Whereas GGMP repeats are present in PfHsp70-1, they are missing in DnaK [ 29 ].…”

“…Whereas GGMP repeats are present in PfHsp70-1, they are missing in DnaK [ 29 ]. One of the unique functional features of PfHsp70-1 is that it binds to asparagine enriched peptide substrates [ 13 ]. Interestingly, it is estimated that 30% of P. falciparum proteome is glutamate/asparagine rich and hence prone to misfolding and aggregation [ 31 , 32 ].…”