Heat shock protein 70 (Hsp70) molecular chaperones are involved in protein folding. Plasmodium falciparum
Hsp70-1 (PfHsp70-1) is a cytosol-localized molecular chaperone that is essential for survival of the malaria parasite [1
]. PfHsp70-1 has been proposed as a prospective antimalarial drug target [2
]. Furthermore, PfHsp70-1 is implicated in antimalarial drug resistance, making its inhibition using antimalarial drug combinations promising [5
]. Although some compounds that inhibit PfHsp70-1 reportedly target the parasitic protein, exhibiting minimum effects on human Hsp70 [4
], the unique structure-function features of this protein remain to be fully explored.
Structurally, Hsp70 consists of two functional domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal SBD [6
]. The NBD of Hsp70 binds to ATP, hydrolyzing it to ADP [7
]. On the other hand, the SBD binds to the peptide substrate. The SBD of Hsp70 is sub-divided into α and β-subdomains. The Hsp70 SBDβ makes direct contact with the peptide substrates while the SBDα serves as a lid enclosing the bound substrate [8
]. Hsp70 exhibits high affinity for substrate in its ADP-bound state and releases it upon binding to ATP [6
]. Therefore, nucleotide binding regulates substrate binding and release by the Hsp70 chaperone. Hsp70 possesses weak basal ATPase activity and hence relies on a co-chaperone, Hsp40, which stimulates its ATPase activity [9
]. In addition, Hsp40 binds to mis-folded proteins first before transferring them to Hsp70 for refolding [10
]. Thus, delivery of substrate to Hsp70 is concomitantly linked to ATP hydrolysis [11
Hsp70 (DnaK) structurally constitutes a canonical Hsp70, characterized by a conserved NBD connected to the SBD via a highly conserved linker motif [12
]. Although DnaK is not essential for E. coli
growth at intermediate temperatures, it is essential at high growth temperatures [13
]. Both PfHsp70-1 and E. coli
DnaK are cytosolic chaperones. PfHsp70-1 and a chimeric protein, KPf (made up of the ATPase domain of DnaK and the SBD of PfHsp70-1), were previously shown to protect E. coli dnaK756
cells (harboring a functionally compromised DnaK) against heat stress [14
]. In addition, PfHsp70-1 has been shown to provide cyto-protection to yeast cells endowed with a defective Hsp70 [15
]. Altogether, this suggests that although Hsp70s are functionally specialized, they also exhibit functional overlaps across species. For example, it was previously reported that PfHsp70-1 possesses higher basal ATPase activity compared to its human and E. coli
Hsp70 homologues [3
]. The unique structure-function features of PfHsp70-1 in comparison to human Hsp70, have spurred interest to target it as part of anti-malarial drug design efforts [3
Hsp40 (PF3D7_1437900) is a co-chaperone of PfHsp70-1 that co-localizes with it to the parasite cytosol [17
]. PfHsp40 is regarded as a member of the so-called type I Hsp40s on account its structural resemblance to E. coli
Hsp40 (DnaJ; [18
]). Both DnaJ and PfHsp40 possess a highly conserved J domain that facilitates cross-talk with Hsp70 [18
]. It has been shown that Hsp40 chimeric proteins with J domains of variable eukaryotic origin cooperated with DnaK to confer cyto-protection to E. coli
cells that were void of endogenous DnaJ [19
]. This suggests that the highly conserved J domain of Hsp40 is capable of modulating the function of Hsp70s of varied species origin. Although functional specificity of Hsp70s across species is generally regarded to be on account of their cooperation with several Hsp40 partners [20
], we still do not understand how such conserved molecules are adapted to their function. It is further believed that of the two domains of Hsp70, it is the less conserved SBD that provides it with functional specificity [7
It has been proposed that nearly 10% of P. falciparum’s
proteome is characterized by prion-like repeats and that at least 30% of the proteome is characterized by glutamate/asparagine rich segments [21
]. For this reason, it is thought that P. falciparum
Hsp70s are adapted to fold and stabilize its mis-folding-prone proteome [23
]. To this end, we previously demonstrated that a P. falciparum
chaperone, PfHsp70-x, which is exported to the parasite-infected red blood cell [26
], exhibits preference for asparagine rich peptides, further suggesting that P. falciparum
Hsp70s are primed to bind mis-folded proteins of the parasite [25
Both PfHsp70-1 and its chimeric product, KPf, have been shown to confer cyto-protection to E. coli
cells harboring functionally compromised DnaK [27
]. This suggests that PfHsp70-1 and KPf exhibit functional overlap with DnaK. On the other hand, PfHsp70-1 and KPf have both been employed to improve the quality and yield of recombinant proteins of plasmodial origin expressed in E. coli
]. This suggests that although PfHsp70-1 and KPf exhibit functional overlap with DnaK, they could be tailored to facilitate folding of proteins of plasmodial origin. For these reasons, PfHsp70-1, DnaK and their chimeric protein, KPf, present a convenient model for studying the functional specificity of PfHsp70-1.
adenosylmethionine decarboxylase (PfAdoMetDC) is an essential protein involved in the biosynthesis of polyamines, making it a potential anti-malarial drug target [29
]. Previously, we demonstrated that recombinant PfAdoMetDC co-expressed in E. coli
with either KPf or PfHsp70-1 exhibited higher enzymatic activity than that co-expressed with supplementary E. coli
GroEL and its cofactor, GroES, constitute a chaperonin of E. coli
that is constituted of a cylindrical complex of two heptameric rings [30
]. Thus, the GroEL/ES system constitutes a cage into which some mis-folded proteins are sequestered to facilitate their folding. It has been proposed that the GroEL/ES cage accommodates substrates of up to 60 kDa in size [31
]. GroEL/ES and DnaK cooperate to facilitate folding of some proteins in E. coli
]. For this reason, we investigated the effect of the three Hsp70s on the folding status of recombinant PfAdoMetDC expressed in E. coli
(DE3) cells. We further expressed each of the Hsp70 along with GroEL towards exploring their combined influence on PfAdoMetDC folding.
Our findings established that all the three Hsp70s exhibited comparable secondary and tertiary structures and they also shared some functional features. However, both PfHsp70-1 and KPf preferentially bound to peptide substrates that were enriched for asparagine residues while the presence of asparagine did not enhance the affinity of DnaK for these peptides. In addition, both PfHsp70-1 and KPf were marginally more stable to heat stress than DnaK. Our findings highlight the importance of the SBD of Hsp70 in stabilizing the conformation of this chaperone and its role in defining the functional specificity of the molecular chaperone. In addition, PfAdoMetDC co-expressed in E. coli with PfHsp70-1 and KPf exhibited similar biophysical features and was better folded than the protein co-produced with supplementary E. coli DnaK. This further demonstrates that the SBD of PfHsp70-1 could be structurally tailored to fold proteins of plasmodial origin.
PfHsp70-1 plays an important role in the survival and development of P. falciparum
, the main agent of malaria. There is evidence that notwithstanding their sequence conservation, Hsp70s exhibit specialized functional features across species. To the best of our knowledge this study for the first time demonstrates that PfHsp70-1 preferentially bound to asparagine enriched peptide substrates in vitro. Furthermore, expression of PfHsp70-1 and KPf in E. coli
improved PfAdoMetDC folding. On the other hand, the enrichment of model Hsp70 peptide substrates with asparagine did not improve their affinity for E. coli
DnaK. In addition, recombinant PfAdoMetDC folding did not benefit from co-production with supplementary DnaK in E. coli
. Our findings do not only demonstrate the unique functional features of PfHsp70-1 relative to DnaK but also highlight the role of the SBD of Hsp70 in the interaction of this protein with nucleotide and the Hsp40 co-chaperone. One of the defining features of cytosolic Hsp70s of parasitic origin is the prominent presence of GGMP repeat motifs located in their C-terminal SBD [1
]. The GGMP motif and other unique residues present in PfHsp70-1 may confer it with unique functional features.
Heterologously expressed forms of DnaK, PfHsp70-1 and their chimeric product, KPf, previously reversed the thermosensitivity of E. coli dnaK756
cells, whose native DnaK is functionally compromised [27
]. The chimera KPf is made up of the NBD of DnaK and shares the same SBD as PfHsp70-1. For this reason, KPf constituted an appropriate tool to explore the unique functional features of PfHsp70-1 relative to E. coli DnaK. Our findings established that KPf exhibits structure-function features that are unique from its parental isotypes. In addition, co-expressing KPf, DnaK, and PfHsp70-1 with recombinant PfAdoMetDC in E. coli
, led to the production of PfAdoMetDC protein with unique secondary and tertiary conformations. The findings demonstrate that although the SBD regulates the functional specificity of PfHsp70-1, the cooperation of both the SBD and the NBD is important for this process. We established that KPf was nearly as stable to heat stress as PfHsp70-1, and that DnaK in turn was less stable than both KPf and PfHsp70-1 (Figure 1
). This suggests that the SBD of PfHsp70-1 which it shared with KPf, conferred stability to both KPf and PfHsp70-1. This is in concert with a previous study [41
] which reported that the SBD of PfHsp70-1 is important for the stability of the protein. As such, the SBD of DnaK may account for its comparatively lower stability to heat stress. We previously observed that the C-terminal EEVN residues of PfHsp70-x (P. falciparum
Hsp70 that is exported to the parasite-infected red blood cell), contributes to the overall stability of the protein [25
]. This further suggests a role of the SBD in regulating Hsp70 stability.
The structural features that KPf shared with PfHsp70-1 confirm the important role of the SBD of Hsp70 in modulating Hsp70 function. However, KPf also possesses key structure-function features that set it apart from PfHsp70-1. KPf exhibited much higher affinity for ATP than DnaK (>200-fold) and its affinity for ATP was at least an order of magnitude higher than of PfHsp70-1 (Table S2
< 0.01). Interestingly, the high affinity for ATP registered by KPf is mirrored by the fact that the chimeric protein was the most conformationally responsive to the presence of ATP (Figure 1
c). It is known that ATP and small molecule inhibitors that bind to the NBD modulate the global conformation of Hsp70 through allostery [27
]. Thus, the enhanced conformational changes that ATP induced on KPf may be on account of the unique NBD–SBD interface of this chimeric protein. Interestingly, both KPf and PfHsp70-1 hydrolyzed ATP more effectively than DnaK (Table S1
< 0.05). It has previously been reported that PfHsp70-1 exhibits higher ATPase activity than Hsp70s of human, bovine and E. coli
]. This suggests that the SBD of Hsp70 plays an important role in regulating its ATPase activity.
PfHsp40 is a P. falciparum
cytosol localized Hsp40 whose structure-function features resemble those of the canonical E. coli
]. PfHsp40 has been shown to stimulate the ATPase activities of cytosol-localized Hsp70s, including PfHsp70-1 and human Hsp70 [17
]. Here we demonstrated that PfHsp40 directly bound to all the three Hsp70s and exhibited comparative affinity for KPf and PfHsp70-1. However, its affinity for DnaK was an order of magnitude lower (Table S1
). Furthermore, PfHsp40 stimulated the ATPase activity of KPf more effectively than it modulated the ATPase activities of either PfHsp70-1 or DnaK (Figure 1
d; Table S1
< 0.01). This finding suggests that the NBD of DnaK and the SBD of PfHsp70-1 constituting the domains of KPf, create a structurally unique NBD–SBD interface that promotes efficient hydrolysis of ATP in the presence of PfHsp40. Since the NBD of Hsp70 is highly conserved while its SBD is fairly divergent, the NBD–SBD interface of Hsp70 is regarded as a unique structural entity that regulates its functional specificity [16
All the proteins were capable of self-association (Table 1
). While KPf and PfHsp70-1 exhibited high affinity (nanomolar range in the presence of ATP), DnaK self-association was weaker (micromolar range) under similar conditions. These findings suggest that ATP promoted self-association of the three proteins, and this is in line with a previous independent study which proposed that oligomerization of DnaK is enhanced by ATP [49
]. Notably, both KPf and PfHsp70-1 exhibited comparably higher affinity for self-association than DnaK. It has been proposed that oligomerization of Hsp70 is mediated by the NBD–SBD interface and the linker segment [46
]. Since both KPf and PfHsp70-1 share the same SBD and a highly conserved linker motif [60
], these two subdomains that the two chaperones share may have accounted for their comparably higher propensity to form oligomers than DnaK.
It has been proposed that roughly 10% of P. falciparum
proteome is marked by prion-like repeats and that more than 30% of the parasite proteins are characterized by glutamate/asparagine repeat segments [21
]. Furthermore, a previous study we conducted demonstrated that the red blood cell exported parasite Hsp70, PfHsp70-x, preferentially binds to peptides enriched with asparagine residues in vitro [25
]. For this reason, we explored the substrate binding preferences of PfHsp70-1 relative to KPf and DnaK.
As expected, in the presence of ADP, DnaK displayed high affinity (nanomolar range) for its model substrate, NRLLTG (p
= 0.05). However, affinity of DnaK for the L–N substitution version of this peptide (NRNNTG) led to a drop in affinity (micromolar range). It is known that DnaK prefers peptides enriched in hydrophobic residues [10
]. Similarly, the enrichment of the other two the peptides, ALLLMYRR and GFRVVLMYRF, with asparagine residues, did not enhance DnaK’s affinity for the peptides in the presence of ADP. On the other hand, introduction of asparagine residues led both KPf and PfHsp70-1 to bind the peptide NRNNTG with higher affinity (nanomolar range) than they displayed for NRLLTG (Figure 2
; Figure S2
= 0.05). In addition, PfHsp70-1 bound with higher affinity to peptides GFRNNNMYR and ANNNMYRR than it exhibited for GFRVVLMYRF and ALLLMYRR (Figure S2
< 0.05). However, KPf did not exhibit enhanced affinity for the asparagine enriched forms of these two peptides. Overall, the findings demonstrate that the SBD of PfHsp70-1 is biased towards asparagine rich peptides. In addition, the data suggest a role for the ATPase domain in regulating the functional specificity of the SBD.
We previously demonstrated that PfAdoMetDC co-produced with supplementary DnaJ–DnaK was less active than protein co-produced with DnaJ-KPf / PfHsp70-1 [24
]. In the current study, we sought to establish whether co-expression of PfAdoMetDC with the various chaperone sets variably modulates its folded status. To this end, we purified recombinant PfAdoMetDC co-produced with the various chaperones we employed here and subjected it to SEC, CD and fluorescence spectrometric analyses. Our SEC analysis suggested that PfAdoMetDC that was co-expressed with DnaK / KPf / PfHsp70-1 + DnaJ + GroEL was more compact than the protein expressed in the presence of only DnaJ-DnaK / KPf / PfHsp70-1 (Figure 3
). Thus, supplementary GroEL appeared to marshal PfAdoMetDC folding towards a more compact conformation (Figure 3
). Interestingly, the elution profile of PfAdoMetDC expressed in the absence of supplementary chaperones eluted at nearly the same retention time as the protein co-expressed with the respective supplementary Hsp70 plus GroEL. The finding further suggests that the presence of supplementary GroEL modulated PfAdoMetDC to fold in a unique fashion.
The CD spectroscopic analysis confirmed that PfAdoMetDC is characterized by a dominant ß-sheet fold as previously reported [34
]. While the co-expression of PfAdoMetDC with DnaJ-KPf/PfHsp70-1 restored the conformation of the protein, co-production of the protein with DnaJ-DnaK led to partial loss of the ß-sheet fold of the protein (Figure 3
). This suggests that DnaK did not support PfAdoMetDC folding. However, combining GroEL with DnaJ-DnaK led to restoration of the ß-conformation of PfAdoMetDC (Figure 3
). While the introduction of GroEL appears to have modulated the fold of PfAdoMetDC co-produced with DnaK, it did not alter the apparent secondary structural fold of PfAdoMetDC co-produced with KPf/PfHsp70-1. This seems to suggest that the presence of KPf and PfHsp70-1 led PfAdoMetDC to a fully folded status. Subsequent analyses of PfAdoMetDC by ANS and the tryptophan/tyrosine fluorescence data corroborated that DnaK confounded PfAdoMetDC folding and that both KPf and PfHsp70-1 were more effective in facilitating its folding process (Figure 4
). Furthermore, based on CD-spectrometry, SEC analysis, and intrinsic fluorescence analyses (Figure 4
; Figure S3
), GroEL facilitated PfAdoMetDC to overcome the barriers presented by DnaK in its folding pathway. However, ANS-fluorescence analysis revealed that although GroEL enhanced folding of PfAdoMetDC, the slight blue shift of the spectrum generated by the protein suggests that GroEL may not have fully folded PfAdoMetDC compared to the quality of protein co-produced with either KPf or PfHsp70-1.
The folded status of PfAdoMetDC as noted using CD and fluorescence spectrometric assays was validated by repeating the assays in the presence of the PfAdoMetDC substrate, SAM. The inclusion of SAM did not influence the observed conformation of PfAdoMetDC. Overall, the findings demonstrate that the folding fate of PfAdoMetDC was dependent on the supplementary Hsp70 with which it was co-expressed in E. coli. In addition, the data demonstrated that GroEL salvaged PfAdoMetDC that battled to fold in the presence of supplementary DnaK.