Abstract:
Hsp70 is a prominent molecular chaperone. Structurally, Hsp70 is composed of two domains,
C-terminus substrate binding domain (SBD) and nucleotide binding domain (NBD). The NBD
of Hsp70 is responsible for its ATPase activity. Some Hsp70s of parasites particularly those of
apicomplexa are marked by GGMP residues. For example, Plasmodium falciparum Hsp70-1
(PfHsp70-1) which occurs in the cytosol and nucleus harbors seven GGMP repeats on the C terminus upstream of the EEVD motif. The function of GGMP residues of Hsp70 is largely
unclear but they were recently reported to be involved in substrate and co-chaperone binding.
Therefore, the main aim of this study was to investigate the role of the GGMP residues of
Hsp70s using E. coli Hsp70 (DnaK) and chimeric protein, KPf as models. Chimeric protein
KPf is made up of NBD of DnaK and SBD of PfHsp70-1. E. coli DnaK lacks the GGMP
residues that are present in PfHsp70-1. DnaK-G (DnaK modified to include GGMP residues)
was created to elucidate the function of these residues. Exogenously expressed KPf and DnaK
are known to reverse the thermosensitivity of E. coli dnaK756 cells. The native DnaK of E.
coli dnaK756 cells is functionally compromised making this strain heat sensitive. KPf617-647
and KPfΔG mutants were previously created by conservative substitution and deleting the
GGMP residues of KPf, respectively. Circular dichroism and tryptophan fluorescence
highlighted that the insertion of GGMP residues did not drastically change the secondary and
tertiary structure of DnaK. Cytoprotection of E. coli dnaK756 cells could not be recovered
when cells were heterologous expressing DnaK-G. DnaK recovered the cytoprotection of the
same cells as expected. Furthermore, mutations on GGMP residues adversely impacted the
chaperone function of KPf with respect to cytoprotection of E. coli dnaK756 cells. On the other
hand, the absence of the GGMP residues results in KPf losing its chaperone function. This
suggests that the SBD of PfHsp70-1 requires the GGMP residues to function, whereas E. coli
DnaK does not require these residues. It was further noted that insertion of the GGMP residues
into DnaK led to toxicity in E. coli dnaK103 cells under permissive growth temperature. A
proteomic study conducted to establish the functional deficiencies of DnaK-G established that
the presence of the GGMP residues compromised the capability of DnaK-G to bind some
substrates. Altogether the findings suggest that GGMP repeats account for the specialized
function of PfHsp70-1 in distinction to that of E. coli DnaK.
