Abstract:
The main malaria agent, Plasmodium falciparum, exhibits a complex life cycle. The parasite initially develops in a poikilothermic mosquito vector before it is subsequently transmitted to a homeothermic human host. As such, the parasite depends on heat shock proteins (Hsps) for maintenance of proteostasis under the physiologically diverse conditions characterising its life cycle. Some of the parasite Hsp70 family members, amongst them, PfHsp70-1 and PfHsp70-z, are essential for parasite survival. PfHsp70-1 is a canonical Hsp70, which closely resembles Escherichia coli Hsp70, DnaK. On the other hand, PfHsp70-z is a non-canonical Hsp70 which belongs to the Hsp110 subfamily. Hsp70s exhibit a highly conserved structural architecture characterized by an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD) adjoined by a linker motif. Although canonical Hsp70 linkers are highly conserved, Hsp110 linkers are less conserved. Moreover, sequence analysis data revealed that PfHsp70-z possesses a highly charged linker motif that is markedly different from that of the most studied Hsp110, Saccharomyces cerevisiae Sse1. To date, the function of the linkers of Hsp110s and PfHsp70-1 largely remains unknown. By creating linker swap versions of PfHsp70-1 and PfHsp70-z, this study sought to elucidate the roles of the linkers of the two chaperones in modulating their functions. A DnaK mutant harbouring a PfHsp70-z linker insertion was also characterized towards further elucidating the function of the PfHsp70-z linker. In silico and biophysical characterization revealed structural variations induced by linker mutations. It was established that the linker of PfHsp70-z confers improved stability to protein structure. On the other hand, the insertion of the PfHsp70-z linker into PfHsp70-1 and DnaK compromised the perturbation of the conformations of these two canonical Hsp70s in response to nucleotide binding. Additionally, the PfHsp70-1 linker induced a 3-fold increase in basal ATPase activity of PfHsp70-z. However, the PfHsp70-z linker reduced PfHsp70-1 basal ATPase activity by half. The PfHsp70-z linker also reduced the affinity of PfHsp70-1 for PfHsp40. This possibly accounted for the lack of PfHsp40-induced stimulation of ATPase activity in the PfHsp70-1 linker mutant. Moreover, there was a marked reduction in the refolding efficiency of PfHsp701 and PfHsp70-z linker mutants, respectively. Taken together, this study provided evidence that PfHsp70-1 and PfHsp70-z linkers dictate the structural conformation and functional specifications of the respective proteins. The unique linker PfHsp70-z appears to regulate the chaperone function of this medically important protein.
