Structural-functional diversity of malaria parasite’s PfHSP70-1 and PfHSP40 chaperone pair gives an edge over human orthologs in chaperone-assisted protein folding

Plasmodium falciparum, the human malaria parasite harbors a metastable proteome which is vulnerable to proteotoxic stress conditions encountered during its lifecycle. How parasite’s chaperone machinery is able to maintain its aggregation-prone proteome in functional state, is poorly understood. As HSP70-40 system forms the central hub in cellular proteostasis, we investigated the protein folding capacity of PfHSP70-1 and PfHSP40 chaperone pair and compared it with human orthologs (HSPA1A and DNAJA1). Despite structural similarity, we observed that parasite chaperones and their human orthologs exhibit striking differences in conformational dynamics. Comprehensive biochemical investigations revealed that PfHSP70-1 and PfHSP40 chaperone pair has better protein folding, aggregation inhibition and oligomer remodeling and disaggregase activities than their human orthologs. Chaperone-swapping experiments suggest that PfHSP40 can also efficiently cooperate with human HSP70 to facilitate folding of clientsubstrate. SPR-derived kinetic parameters reveal that PfHSP40 has higher binding affinity towards unfolded substrate than DNAJA1. Interestingly, the observed slow dissociation rate of PfHSP40-substrate interaction allows PfHSP40 to maintain substrate in folding-competent state to minimize its misfolding. Structural investigation through SAXS gave insights into the conformational architecture of PfHSP70-1 (monomer), PfHSP40 (dimer) and their complex. Overall, our data suggests that parasite has evolved functionally diverged and efficient chaperone machinery which allows human malaria parasite to survive in hostile conditions. The distinct allosteric landscapes and interaction kinetics of plasmodial chaperones open avenues for exploration of small-molecule based antimalarial interventions. D ow naded rom http://pndpress.com /bchem j/article-oi/10.1042/BC J20200434/6/bcj-2020-0434.pdf by gest on 20 Sptem er 2020 Bchem al Jornal. This is an Acepted M ancript. ou re encuraged to se he Vrsion of R eord tat, w en puished, w ill relace his vesion. he m st up-tote-version is avilable at https://drg/10.1042/BC J200434


Introduction
Human malaria parasite encounters frequent environmental fluctuations (pH, nutrients, temperature, oxygen) and immune threats as it homes different tissues; and transits through warm-blooded host (human for asexual cycle) and cold blooded vector (mosquito for sexual cycle) [1]. Amongst the malaria causing Plasmodium spp., P. falciparum has acquired striking proteome features that are distinct from other species. Its proteome harbors low complexity regions which have high frequency of asparagine and glutamines homorepeats [2][3][4]. Homorepeats impart prion-like features or disorder which allows proteins to undergo dynamic conformational transitions for protein-protein interactions [5,6]. However, this structural-functional advantage of a proteome enriched with disordered domains has a trade-off associated with thermodynamic and kinetic constrains in protein folding [7]. Hence, acquisition of homorepeats in a proteome is inter-linked with co-acquisition of a robust proteostasis machinery [8,9]. This machinery involves close cooperation between protein folding and degradation processes to limit cellular protein misfolding and/or coordinate efficient disaggregation or degradation of misfolded proteins.
Phylogenetic analysis of P. falciparum has revealed that its proteostasis machinery has significantly diverged from its human orthologs, and few components even exhibit species-specific sequence variations which may promote auxiliary interactions to give survival advantage to the parasite during hostile microenvironments [10,11]. During protein synthesis in Plasmodium, frequent environmental perturbations may interfere with correct folding of nascent polypeptide, its targeting to subcellular compartments and export of parasite's cargo. The cellular chaperones form the first-line of defense by interacting with nascent polypeptide to ensure that it has attained its functional state and is properly targeted to the destined cellular organelle. The chaperone network primarily includes ribosome-bound chaperones, sHSPs, HSP40, HSP70, HSP60, HSP90 and HSP110 [12][13][14][15][16][17]. In the cellular protein folding cascade, HSP70-HSP40 forms the central hub which facilitates co-and post-translational folding of nascent polypeptides [12,13]. It also interacts with the disaggregase and degradation machinery to coordinate refolding of misfolded polypeptides or efficient removal of terminally aggregated species [18][19][20][21]. HSP70 family is ubiquitous and highly conserved in different organisms, while its co-chaperone HSP40 family comprise of structurally and functionally diverse proteins which drive substrate specificity to HSP70-mediated protein folding and disaggregation [22,23]. Plasmodial HSP70s (4 isoforms) and HSP40s (45 isoforms) are localized in distinct sub-cellular compartments (cytosol, nucleus, apicoplast, mitochondrion, ER) [24][25][26][27][28]. They are even exported into human RBC to facilitate trafficking of parasite cargo proteins to sequester nutrients from host, export of virulence factors (PfEMP1) via transient cellular organelles onto RBC surface and modulate the repertoire of client proteins for immune evasion [29][30][31][32]. Cooperation of HSP70-HSP40 has been extensively investigated for bacterial, yeast and human orthologs [33][34][35][36][37]. Studies have revealed key variations in the interaction interfaces between HSP70-HSP40 and client proteins; and this fine tunes HSP70-HSP40 activity in different organisms [20,[37][38][39]. Such biochemical and structural investigations for P. falciparum chaperones are lacking due to challenges associated with poor protein expression related to codon bias, solubility, concentration-dependent oligomer formation and proteolysis issues. Till date, biochemical studies for parasite chaperones have been limited to ATPase activity and suppression of aggregation [40][41][42][43][44]. Plasmodial HSP70-HSP40 have also been suggested to be potential drug targets [45,46]. However, this exploration requires thorough structural-functional characterization and comparison with human orthologs to minimize off-target effects in antimalarial interventions. In this study, we have performed comprehensive biochemical investigations (conformational dynamics, refolding, aggregation inhibition, oligomer remodeling, disaggregase activity and interaction kinetics) for plasmodial HSP70-40 (PfHSP70-1, type-I PfHSP40) machinery and compared it with their human orthologs. In addition, we provide the insights into the shape and conformation of PfHSP70-1, PfHSP40 and their complex. Our data suggests that parasite has evolved an efficient PfHSP70-1 and PfHSP40 system which supports parasite survival in human host. These findings open avenues for rational small-molecule based SAR for disruption of critical parasite chaperone interactions for antimalarial interventions.
Purified protein was loaded on S200 increase 10/300 (GE Healthcare), pre-equilibrated with buffer (50 mM Tris pH 7.5, 5 mM MgCl 2 , 500 mM KCl, 2 % glycerol, 1 mM β-mercaptoethanol). Elution peak corresponding to dimer was collected and supplemented with 10% glycerol for storage in -80 °C. monomer was used to raise polyclonal antibody. Purified luciferase protein (500 µg/mL) was mixed with Fruend's complete adjuvant (1:1) which was injected subcutaneously in to NZW female rabbit followed with booster 1 after 28 days of immunization. Booster 2 was given 14 days after the first booster dose.
The booster dose is prepared with luciferase protein (500 µg/mL) mixed with Fruend's incomplete adjuvant. Blood was collected after ten days of second booster. Antisera was collected by processing the blood. Purified luciferase protein was run on SDS PAGE and transferred on to nitrocellulose membrane followed by blocking the membrane with 5% skimmed milk for 1 h at room temperature. Membrane was This elution step was repeated three times. 10 % glycerol was added in eluted polyclonal antibody and stored at -80 °C. The specificity of luciferase antibody was compared with pre-immune sera against luciferase protein.

Probing conformational dynamics in human and parasite chaperones
Limited proteolysis experiments (using 2 µM trypsin, Sigma T6567) were performed with human (40 µM HSPA1A) and parasite chaperone (40 µM PfHSP70-1) in absence or presence of nucleotide (apo) and/or NR-peptide. Prior to trypsin digestion, parasite and human HSP70 was incubated with 5 mM ATP (Sigma

ATPase Assay
ATPase activity of parasite and human HSP70 was determined through Enzcheck phosphate assay kit (E6646). For a total reaction volume of 400 µl, parasite and human HSP70 (1 µM) and HSP40 (4 µM) were incubated in buffer X (50 mM Tris pH 7.5, 12 mM MgCl 2 , 50 mM KCl, 2 mM β-mercaptoethanol) for 15 mins at 25 °C and 37 °C. This was followed by addition of 80 µl of 2-amino-6-mercapto-7methylpurine riboside (MESG) substrate and 4 µl of purine nucleoside phosphorylase (PNP) (provided in the kit) with additional incubation for 15 mins at 25 °C and 37 °C. For control reaction, above mentioned procedure was followed with BSA (5 µM). Reactions were started by adding 2 mM ATP (Sigma A2383) and read-out for ATP hydrolysis was monitored at Abs360 nm using UV-vis spectrometer (Jasco V750) at 25 °C and 37 °C.

Foldase, holdase and oligomer remodeling activities
Foldase activity: Recombinant luciferase (2 mg/mL) was precipitated by adding five volumes acetone and centrifuge at 10000 rpm for 20 mins at 4 °C as described previously [47]. Pellet was resuspended in   Biaevaluation software was used to process the sensograms. The k on (association rate) and k off (dissociation rate) were obtained through bivalent fitting. The second association rate constant (kon2) is calculated through kon 2 (M -1 s -1 ) = kon 2 (RU -1 s -1 )×Mr×100 (where Mr is molecular weight). Molecular weight of PfHSP40 (88 kDa) and DNAJA1 (85 kDa) was determined through size exclusion chromatography by using protein standards (GE Healthcare 28-4038-42).

Small angle x-ray scattering (SAXS)
Purified PfHSP70-1 was incubated with 5 mM ADP and 100 µM NR-peptide, and concentrated through 3 PfHSP70-1 and PfHSP40 were incubated with 5 mM ADP and 100 µM NR-peptide. The sample was concentrated upto 4 mg/mL. SAXS data was collected using Anton Paar SAXS space instrument at CSIR-Central Drug Research Institute (Lucknow, India) using a MYTHEN2 R 1K detector at a sample-detector distance of 0.3 m and at a wavelength of 0.154 nm. Scattering was measured at 10 °C. Two successive 1800 sec frames were collected. The data was normalized to the intensity of the transmitted beam and radially averaged. The scattering of the buffer was subtracted from solution scattering of samples. The data was processed by PRIMUSQT. The forward scattering intensity I(0) and radius of gyration R(g) were estimated using AUTOGNOM which was also used to evaluate the molecular size by plotting pairdistance distribution functions (PDDF). P(r) of scattering data is the representation in real space and reflects the particle's shape. From SAXS scattering profile, 10 independent ab initio bead models were generated through DAMMIF. The average bead model was superimposed with homology models through SUPCOMB.

Conformational dynamics of human and parasite chaperones
We performed limited proteolysis of human and plasmodial chaperones (HSP70 and HSP40) to understand whether sequence diversity translates into differences in conformational dynamics. HSP70 is an allosteric chaperone that undergoes nucleotide-dependent conformational dynamics into an open state in presence of ATP and closed state in presence of ADP. HSP70 has high affinity towards substrate in ADP-bound state, whereas in ATP-bound state, HSP70 releases the substrate [55,57,61,62]. In apo-state (without nucleotide or peptide), human HSP70 (HSPA1A) adopts ensemble conformations which are highly protease sensitive due to exposure of proteolytically labile regions to protease digestion ( Figure 1).
However, in nucleotide-bound state (ADP or ATP), HSPA1A exhibits protease resistance [63]. The relative orientation of HSPA1A subdomains is different in ATP and ADP-bound state which give rise to subtle differences in protease digestion profile. In ATP-bound state, nucleotide binding domain (NBD) docks onto substrate binding domain (SBDβ) which intimately packs the inter-domain linker between these two domains and opens the cleft between SBDβ and SBDα (docked or open conformation) [57].
While in the ADP-bound state, NBD is in undocked state with solvent-exposed inter-domain linker and SBDα is folded onto SBDβ (undocked or closed conformation). In presence of ADP and peptide, SBD has high affinity towards peptide which favors undocked or closed conformation and thus exposes protease sensitive regions. Interestingly, such dramatic differences in proteolytic profile were not observed for PfHSP70-1 in apo-state, nucleotide-bound state or in presence of peptide. This suggests that the relative orientation of subdomains, flexibility of inter-domain linker and loops affects the allosteric landscape of PfHSP70-1 (Figure 1). Similarly, limited proteolysis experiment was performed with human and parasite HSP40 in absence and presence of peptide. This HSP40-interacting peptide is derived from conserved C-terminal amino acid stretch (8-mer) of HSP70 [64,65]. In this experiment, we observed that human HSP40 (DNAJA1) showed higher protease sensitivity in comparison to parasite HSP40 (PfHSP40) both in apo and peptide-bound state ( Figure 2). Digestion profile obtained for DNAJA1 suggests that the likely cleavage site is in G/F region which serves as a flexible linker between the Nterminus J-domain and C-terminus peptide binding domain. In PfHSP40, the length and sequence of G/F region is different from DNAJA1 (Supplementary Figure S2B), which may contribute to differences in their conformational dynamics. We also performed CD melting studies to understand the thermal stability of human and parasite chaperones. Although, HSP70 and HSP40 orthologs had similar thermal stability, but they exhibited difference in their denaturation profile suggesting that they undergo distinct unfolding transitions (Supplementary Figure S3).

Differences in protein folding capacity of human and parasite chaperones
We wanted to compare the protein folding efficiency of human and plasmodial HSP70-40 chaperone pair.
HSP40 stimulates ATPase activity of HSP70 by binding to its NBD and facilitates HSP70-mediated protein folding [66]. We observed that both human and plasmodial HSP40 stimulated the ATPase activity of their cognate HSP70 to a similar extent (Supplementary Figure S4 and Supplementary Table S2). We further probed into the foldase (refolding) and holdase (prevention of aggregation) activities of plasmodial and human HSP70-HSP40 at 25 °C. Plasmodial chaperones PfHSP70-1 and PfHSP40 exhibited better refolding of chemically denatured substrate than their human orthologs (HSPA1A and DNAJA1) ( Figure 3A). In addition, plasmodial chaperones also exhibited better holdase activity for thermally denatured substrate ( Figure 3B). Holdase activity suggest that plasmodial HSP40 (PfHSP40) has better ability to maintain the polypeptide in folding-competent state in comparison to its human ortholog (DNAJA1) ( Figure 3B). PfHSP40 could also mediate a concentration-dependent increase in protein folding capacity, whereas, its human othrolog (DNAJA1) had moderate suppressive effect on protein folding yields at higher concentrations (Supplementary Figure S5). The foldase and holdase experiments were also performed at mildly denaturing temperature. At 37 °C, luciferase is vulnerable to misfolding and aggregation which resulted in low refolding yields for both plasmodial and human chaperones. Nevertheless, plasmodial chaperones exhibited better folding efficiency (especially holdase activity) than human chaperones at physiological temperature (Supplementary Figure S6). To confirm whether the observed better protein folding capacity of plasmodial chaperones is substrate-specific, we monitored refolding of heat denatured malate dehydrogenase (MDH). Plasmodial chaperones (PfHSP70-1 and PfHSP40) mediated better refolding of denatured MDH (Supplementary Figure S7). We also evaluated the oligomer remodeling ability of plasmodial and human chaperone pair by using limited proteolysis assay. Luciferase monomer is trypsin sensitive, whereas its oligomer is trypsin resistant.
PfHSP70-1 and PfHSP40 system remodels the oligomer, making it sensitive to protease digestion ( Figure   4A). This remodeling probably helps them to attain enzymatically active conformation resulting in concomitant restoration of luciferase activity ( Figure 4B). Similarly, better disaggregation ability was observed for plasmodial chaperones (PfHSP70-1 and PfHSP40) over human chaperones (HSPA1A and DNAJA1) (Supplementary Figure S8). Sensograms were analyzed through various predefined fitting models, however satisfactory fitting results were obtained through bivalent interaction model ( Figure 6). This involves two-step concerted binding, wherein the binding sites can have variable binding affinities [67] and this binding model is in agreement with the recent solution structure of type-I HSP40-unfolded substrate complex [38]. Kinetics data revealed that the association (k on1 ) rate for substrate binding was moderately higher for PfHSP40, but dissociation (k off1 ) rate was significantly lower than human ortholog (Table 1). k on1 and k off1 rates together contribute to the higher binding affinity (equilibrium association constant, K A1 ) of PfHSP40 towards unfolded substrate in comparison to DNAJA1. The second binding site serves as low-binding affinity region and only modest difference in K A2 was observed for plasmodial and human HSP40. Overall, SPR data corroborates with refolding experiments that PfHSP40 efficiently binds to unfolded polypeptide and maintains it in folding-competent state to prevent its misfolding or aggregation.

SAXS analysis of plasmodial HSP70, HSP40 and their complex
We performed SAXS experiments to investigate the shape and conformational structure of PfHSP70-1, PfHSP40 and their complex. Under our experimental set-up, satisfactory solution scattering for plasmodial chaperones was obtained at high concentrations (7-9 mg/mL). For PfHSP70-1, we could not record solution scattering in apo and ATP-bound state due to concentration-dependent aggregation (at conc >5 mg/mL). Previous studies have also shown that HSP70 is prone to form dimers and multimers [68]. SAXS experiment was performed for PfHSP70-1 in presence of ADP and NR-peptide. The scattering profile was analyzed through Guinier plot to check the quality of sample. Guinier plot for PfHSP70-1 followed a linear profile suggesting the monodispersity of sample (Supplementary Figure S9).  Table 2). In SAXS experiment with PfHSP40, the Guinier plot suggested that the protein sample is monodisperse and does not contain any  [38,66,70,71]. In presence of ADP and peptide, the parasite chaperone complex comprises of PfHSP70-1 (monomer) and PfHSP40 (dimer) corresponding to molecular weight of 166 kDa ( Table 2). DAMMIF was used to generate ab initio model, but the fitting onto solution scattering was not good due to poor buffer subtraction. We used ClusPro to build in silico model of PfHSP70-1 and PfHSP40 complex which was superimposed on the bead model generated through DAMMIF. The 3-D reconstruction with bead model gave hints into the overall architecture of the PfHSP70-1 and PfHSP40 complex.

Discussion
In this study, we highlight the fundamental differences in dynamics and chaperoning activity of malaria parasite chaperones (PfHSP70-1 and PfHSP40) and their human orthologs. Both PfHSP70-1 and its cytosolic co-chaperone (PfHSP40) are constitutively expressed during malaria parasite's asexual cycle in human and sexual cycle in mosquito (Supplementary Figure S1A-B). These chaperones are also induced under stress conditions [40,51], hence they participate both in house-keeping and stress-response related processes. PfHSP70-1 exhibits higher similarity to HSPA1A (inducible) over HSPA8 (constitutive) orthologs in human. Similarly, PfHSP40 has higher similarity with DNAJA1 amongst DNAJ protein family. Despite structural similarity, plasmodial and human HSP70-40 homologs exhibit striking differences in their conformational dynamics (Figure 1-2). The observed differences in conformational flexibility of PfHSP70-1 and HSPA1A suggest (i) differences in the number of exposed protease sensitive sites, (ii) variable length or flexibility of inter-domain linker and loops, and (iii) differences in the interaction interface of NBD-SBDβ and SBDβ-SBDα substrate-binding cleft. The conformational heterogeneity in docking/undocking of NBD-SBDβ and opening/closing of SBDβ-SBDα cleft dramatically affect the allosteric landscape of plasmodial and human HSP70 (Figure 1). A similar study showed allosteric differences between eukaryotic HSP70 (HSPA1A, HSC70) and the bacterial homolog Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200434/892626/bcj-2020-0434.pdf by guest on 20 September 2020 (DnaK) [63]. A recent study showed that mutations in the SBDβ-SBDα interface in HSPA1A affects the inter-domain communication between NBD and SBDβ [69]. In case of of plasmodial (PfHSP40) and human HSP40 (DNAJA1), variations in conformational dynamics may arise due to the differences in the flexibility of G/F region and relative orientation of domains ( Figure 2).
The physiological advantage of an efficient HSP70-40 system is associated with cost-benefit of refolding and disaggregation over an energetically-intensive process like re-synthesis of protein. The observed differences in HSP70-mediated protein folding in human and parasite is driven by their respective HSP40 co-chaperone. Sequence comparison of DNAJA1 and PfHSP40 shows that parasite HSP40 has diverged peptide binding domains hinting towards differences in their interaction with unfolded substrate (Supplementary Figure S2B). SPR experiment derived kinetic parameters explains why plasmodial HSP40 (PfHSP40) is better than its human ortholog. Higher association rate and slower dissociation rate allows PfHSP40 to bind unfolded substrate and maintain it in folding-competent state to minimize its misfolding or aggregation (Figure 3 and 6, Table 1). Our data suggests that the functional diversity of PfHSP40 (type-I cytosolic HSP40) gives an edge over its human ortholog (DNAJA1) in driving HSP70- The functional diversity of HSP40 interaction with HSP70 and substrate is influenced by the sequence variation of in its peptide binding domains [37,38,73]. For instance, yeast HSP40s (Ydj1 and Sis1) are suggested to have differences in binding to HSP70 and client-substrate. Ydj1 can engage both clientsubstrate and HSP70, whereas, Sis1 cannot interact with them simultaneously due to overlapping binding sites [38]. Similarly human cytosolic type-I HSP40s (DNAJA1 and DNAJA2) show distinct differences in substrate binding and release [37]. At high concentrations, DNAJA1 impairs the protein refolding yields due to competing reactions for binding to HSP70 and substrate [67]. On similar lines, we observed that high concentration of DNAJA1 did not enhance the protein refolding yields and rather had modest suppressive effect. Whereas, its plasmodial homolog enhanced protein folding yields in concentrationdependent manner (Supplementary Figure S5). We also probed into the structural architecture of parasite chaperones (PfHSP70-1, PfHSP40 and their complex). Till date, high resolution structures are only available for domains of prokaryotic and eukaryotic HSP70 and HSP40 homologs [60-62, 74, 75]; and not for their full length proteins.
Conformational dynamics and transient nature of chaperone and co-chaperone interaction makes structural characterization of HSP70-HSP40 complex technically challenging. Using SAXS, we obtained insights into the structural properties of PfHSP70-1, PfHSP40 and their complex. SAXS data for PfHSP70-1 shows that it adopts an extended conformation in presence of ADP and peptide (Figure 7). This correlates with SAXS data of Leishmania mitochondrial HSP70 [76]. Similarly, solution scattering for PfHSP40 indicates that it exist as a dimer as reported previously for bacterial HSP40 [77]. We also collected SAXS data for PfHSP70-1-40 complex. Although, the bead model generated gave poor fitting to solution scattering, nevertheless, it gave hints into the overall architecture of complex comprising of PfHSP70-1 and PfHSP40 dimer. Despite sequence variations, we observe an evolutionary conserved electrostatic interaction between J-domain of PfHSP40 and NBD of PfHSP70-1 which correlates with the previous data for bacterial DnaK-DnaJ complex (Figure 7). This interaction could be driven by helix II in J-domain and IIA lobe in NBD, as observed in case of DnaK-DnaJ complex [70]. MD simulation study on DnaK-DnaJ complex suggest that conformational flexibility of J-domain facilitates additional interactions with DnaK at inter-domain linker and SBDβ [78]. Similar MD study with PfHSP70-x and exported type-II HSP40 (PFA0660w) shows that J-domain makes stable interactions with NBD and SBD of PfHSP70-x [79] which corroborates with recent structural analysis of complex formed by NBD of PfHSP70-x and J domain of PFA0660w [80]. This MD study also predicted an additional interaction site wherein, the G/F region of PFA0660w makes contact with SBDβ of PfHSP70-x [79]. A recent smFRET study for human HSP70 (hHSP70) and Hdj1 (type-II HSP40) chaperone pair suggests that Hdj1 dimer induces dimerization of hHSP70 to form a hetero-tetramer complex [81]. Transient dimerization of HSP70 for efficient interaction with HSP40 is also reported for bacterial homologs [82]. A NMR based structural characterization of interaction between bacterial HSP40 (ttHSP40) and C-terminal tail of ttHSP70 (15 amino acids long polypeptide) shows that this C-terminal tail makes contact with peptide binding domain II of ttHSP40. However, this interaction interface varies in different eukaryotic HSP40s [38]. Numerous literature observations across species suggest that the conformational heterogeneity of HSP70-HSP40 complex depends upon nucleotide, co-chaperone and substrate [38,66,70,71], leading to functional versatility in their biological roles.
Our biochemical data give hints into how the cytosolic HSP70-40 machinery is chaperoning parasite's metastable proteome against the proteotoxic stress conditions encountered during lifecycle in coldblooded mosquito and warm-blooded human host. Similar investigations are required to understand Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200434/892626/bcj-2020-0434.pdf by guest on 20 September 2020 distinct chaperoning capabilities of organellar and exported plasmodial chaperones. Studies have shown that exported chaperones are involved in trafficking of parasite cargo proteins (in unfolded or partially folded state) into cytosol of human RBCs, where they refold with help of human chaperones and/or exported parasite chaperones [83][84][85]. The transient organelles like maurer's cleft, J-dots, formed in human RBCs upon P. falciparum infection, harbor exported HSP70-40 which mediate the trafficking and assembly of parasite virulence factor on RBC membrane [29,84,86]. This facilitates remodeling of RBC membrane, increases its cytoadherence and prevents the clearance of infected-RBCs by spleen [87].
Human RBCs are e-nucleated and transcriptionally/translationally silent, thus they cannot mount any cellular response to upregulate their chaperones during Plasmodium invasion into RBCs. Whereas, parasite reprograms its cellular machinery and has specific upregulation of its HSPs during infection and febrile episodes [88]. Further, the cellular abundance of parasite's protein folding machinery during infection and disease severity is influenced by the growth rate or metabolic state of parasite and host immune response [89,90]. Therefore, a systematic molecular investigation of parasite's protein folding machinery will provide insights into their versatile roles during progression of infection. And          1.09×10 2 Kinetic constants were determined through SPR experiments. Human HSP40 (DNAJA1) and plasmodial HSP40 (PfHSP40) were used as ligand and unfolded luciferase was used as analyte. Sensograms were fitted with bivalent fitting to obtain rate constants. Equilibrium association constant (K A ) is the ratio of k on /k off . Table 2. SAXS analysis of plasmodial HSP40, HSP70 and their complex a Experimental MW determined through SEC (S200 increase 10/300); b Stokes radius is the hydrodynamic radius determined through SEC. c,d I(0) is the scattering intensity of sample; Rg is radius of gyration; D max is maximum linear dimension; e V porod is the volume of scattering particle obtained through Porod-Debye law; f Fitting of solution scattering of macromolecules with known atomic structure into experimental scattering obtained through SAXS; g ab initio shape determination. Due to poor buffer match, DAMMIF value for complex is very low; h Fitting of high resolution model on experimental scattering to obtain normalized spatial discrepancy (NSD). NSD value is obtained from SUPCOMB. The obtained NSD is >1 because high resolution data is not available for plasmodial HSPs. Homology structures are generated using PDB structures of bacterial orthologs.