Comparative analysis of a chimeric Hsp70 of E. coli and Plasmodium falciparum origin relative to its wild type forms
Sustaining proteostasis is essential for the survival of the cell and altered protein regulation leads to many cellular pathologies. Heat shock proteins (Hsps) are involved in the regulation of the protein quality control. Hsps are a group of molecular chaperones that are upregulated in response to cell stress and some are produced constitutively. The Hsp70 family also known as DnaK in Escherichia coli (E. coli) is the most well-known group of molecular chaperones. Structurally, Hsp70s consist of a nucleotide binding domain (NBD) and a substrate binding domain (SBD) conjugated by a linker sub-domain. ATP binding and hydrolysis is central to the Hsp70 functional cycle. Hsp70s play a role in cytoprotection especially during heat stress in E. coli. Hsp70s from different organisms are thought to exhibit specialized cellular functions. As such E. coli Hsp70 (DnaK) is a molecular chaperone that is central to proteostasis in E. coli. On the other hand, Plasmodium falciparum Hsp70s are structurally amenable to facilitate folding of P. falciparum substrates. The heterologous production of P. falciparum proteins in E. coli towards drug discovery has been a challenge. There is need to develop tools that enhance heterologous expression and proper folding of P. falciparum proteins in an E. coli expression system. To this end, a chimeric Hsp70, KPf consisting of E. coli DnaK NBD and P. falciparum Hsp70-1 (PfHsp70-1) SBD was previously designed. KPf was shown to confer cytoprotection to E. coli DnaK deficient cells that were subjected to heat stress. In this study it was proposed that KPf has an advantage over E. coli DnaK and PfHsp70-1 in its function as a protein folding chaperone. Therefore, the main aim of this study was to characterize the chaperone function of KPf relative to the function of wild type E. coli and P. falciparum Hsp70s. The recombinant forms of KPf, DnaK and PfHsp70-1 proteins were successfully expressed and purified using nickel affinity chromatography. Circular Dichroism (CD) structural study demonstrated that KPf and PfHsp70-1 are predominantly α-helical and are also heat stable. Tertiary structure studies of PfHsp70-1 and KPf using tryptophan fluorescence revealed that both confirmations of recombinant proteins are perturbed by the presence of ATP more than ADP. Interestingly, the substrate binding capabilities of these proteins were comparable both in the absence or presence of nucleotides ATP/ADP. KPf is an independent chaperone, that exhibit nucleotide binding and hydrolysis. The current study has established unique structure-function features of KPf that distinguishes it from its “parental” forms, DnaK and PfHsp70-1.
Heat shock proteins, Chaperone, Kpf, Dnak, PfHsp70-1, PfHsp40
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