Biophysical analysis of Plasmodium falciparum Hsp70-Hsp90 organising protein (PfHop) reveals a monomer that is characterised by folded segments connected by flexible linkers

Stanley Makumire; Tawanda Zininga; Juha Vahokoski; Inari Kursula; Addmore Shonhai

doi:10.1371/journal.pone.0226657

. 2020 Apr 28;15(4):e0226657. doi: 10.1371/journal.pone.0226657

Biophysical analysis of Plasmodium falciparum Hsp70-Hsp90 organising protein (PfHop) reveals a monomer that is characterised by folded segments connected by flexible linkers

Stanley Makumire ¹, Tawanda Zininga ^1,², Juha Vahokoski ³, Inari Kursula ^3,⁴, Addmore Shonhai ^1,^*

Editor: Didier Picard⁵

PMCID: PMC7188212 PMID: 32343703

Abstract

Plasmodium falciparum causes the most lethal form of malaria. The cooperation of heat shock protein (Hsp) 70 and 90 is thought to facilitate folding of select group of cellular proteins that are crucial for cyto-protection and development of the parasites. Hsp70 and Hsp90 are brought into a functional complex that allows substrate exchange by stress inducible protein 1 (STI1), also known as Hsp70-Hsp90 organising protein (Hop). P. falciparum Hop (PfHop) co-localises and occurs in complex with the parasite cytosolic chaperones, PfHsp70-1 and PfHsp90. Here, we characterised the structure of recombinant PfHop using synchrotron radiation circular dichroism (SRCD) and small-angle X-ray scattering. Structurally, PfHop is a monomeric, elongated but folded protein, in agreement with its predicted TPR domain structure. Using SRCD, we established that PfHop is unstable at temperatures higher than 40°C. This suggests that PfHop is less stable at elevated temperatures compared to its functional partner, PfHsp70-1, that is reportedly stable at temperatures as high as 80°C. These findings contribute towards our understanding of the role of the Hop-mediated functional partnership between Hsp70 and Hsp90.

1. Introduction

Heat shock proteins (Hsp) serve primarily as protein folding facilitators. They also participate in several other processes, such as protein transport, assembly/disassembly of protein complexes, protein degradation, amongst others [1]. Their role in the survival and pathogenicity of malaria parasites is increasingly becoming apparent [2, 3, 4]. Plasmodium falciparum is the agent for the most lethal form of malaria. It has been reported that the cytosolic P. falciparum heat shock protein 70–1 (PfHsp70-1) is cyto-protective due to its ability to suppress protein mis-folding and aggregation under stressful conditions [5, 6]. In addition, another cytosolic molecular chaperone, P. falciparum Hsp90 (PfHsp90) is essential [7]. The cooperation of Hsp70 and Hsp90 is known to facilitate folding and function of proteins implicated in cell development, such as steroid hormone receptors and kinases [8, 9].

Stress inducible protein 1 (STI1) was first described in mouse [10], and now also known as Hsp70-Hsp90 organising protein (Hop), acts as a module that allows Hsp70 and Hsp90 to interact stably, thereby facilitating substrate transfer from Hsp70 to Hsp90. Interestingly, Hsp70 and Hsp90 resident in the cytosols of E. coli and yeast as well as their homologs localized to mouse ER were shown to interact directly independent of Hop mediation [11, 12, 13, 14]. Although E. coli lacks a Hop homolog [15] and the same protein is not essential in yeast [16], it has been reported to play an important role in the development of Trypanosoma cruzi and Trypanosoma brucei parasites [17]. This shows that the role of Hop varies across species and developmental stages. In light of the essential roles of the P. falciparum cytosol localised chaperones, PfHsp90 and PfHsp70-1, PfHop has been proposed as a potential antimalarial drug target [18, 19] This is partly because it occurs in complex with the two chaperones, and also exhibits some degree of sequence divergence from the human homolog, thus raising prospects for selective targeting by small molecule inhibitors [18]. Indeed, in some disease models such as cancer [20] and leishmaniasis [21, 22], the essential role of Hop has made it a promising drug target.

Hop is a conserved and stress inducible protein that possesses three tetratricopeptide repeats (TPR): TPR1, TPR2A and TPR2B [10]. Both Hsp70 and Hsp90 interact with Hop via the C-terminal EEVD motif, present in the two molecular chaperones [19, 23]. Hop interacts with Hsp70 and Hsp90 via its TPR1 and TPR2A domains, respectively [23]. While for a long time the role of the TPR2B domain of Hop has remained largely elusive, it is now thought that Hsp70 first binds to the TPR1 domain of Hop before switching to the TPR2B domain to facilitate substrate transfer to Hsp90 [20, 23].

In light of the importance of both PfHsp70-1 and PfHsp90 in the survival of the malaria parasite, there has been growing interest in identifying inhibitors targeting the function of these two molecular chaperones. Compounds that inhibit PfHsp70-1 [24, 25, 26] and PfHsp90 [9, 27] have been identified, and some of them exhibit antiplasmodial activity. Some compounds that target PfHsp90 function reverse parasite resistance to traditional antimalarial drugs, such as chloroquine (reviewed in [28]). We previously described Plasmodium falciparum Hop (PfHop), which co-localises and associates with both PfHsp70-1 and PfHsp90 [18, 19]. While Hop in other organisms, such as yeast and human, has been rather extensively characterised, the structure and function of PfHop remain to be elucidated.

Here, we show that PfHop is a monomeric, elongated but folded protein, which loses most of its secondary structure at temperatures above 40 ºC. We discuss the implications of our findings with respect to the role of PfHop in coordinating the Hsp70-Hsp90 pathway in P. falciparum.

2. Methods and materials

2.1 Materials

Reagents used in this study, unless otherwise stated, were purchased from Merck Chemicals (Darmstadt, Germany), Thermo Scientific (Illinois, USA), Zymo Research (USA), Melford (Suffolk, UK), and Sigma-Aldrich (USA). Nickel NTA resin was purchased from Thermo Scientific (USA). ECL was purchased from (ThermoFisher Scientific, USA). The expression and purification of his-tagged recombinant forms of PfHop was confirmed by Western blotting using anti-His antibodies (Thermo Scientific, USA). Furthermore, rabbit raised anti-PfHop antibodies (Eurogentec, Belgium; [18]) were also used to confirm the presence of recombinant PfHop protein.

2.2 Expression and purification of recombinant PfHop

Recombinant PfHop (PF3D7_1434300) was overexpressed in Escherichia coli XL1 Blue cells and purified by nickel affinity chromatography as previously described [18, 19]. The Ni-affinity purified proteins were extensively dialysed in SnakeSkin dialysis tubing 10 000 MWCO (ThermoFisher Scientific, USA) against buffer A [20 mM Tris-HCl, pH 7.5, 10 mM NaCl, 5% (v/v) glycerol, 0.2 mM Tris-carboxyethyl phosphine (TCEP)]. The protein from Ni-NTA chromatography was used to perform conventional CD spectroscopy and tryptophan fluorescence assays. SRCD and SAXS analyses were performed using protein that was further purified using ion exchange and size exclusion chromatography as follows. The dialysed protein was further purified using anion exchange chromatography using a Tricorn MonoQ 4.6/100 PE column (G.E Healthcare LS, USA). PfHop was eluted by applying buffer B (20 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.2 mM TCEP) to the column using a linear (0.1–1.0 M) NaCl gradient. As the final purification step and to evaluate the oligomeric state of PfHop, size exclusion chromatography was used. Following anion exchange, fractions containing pure PfHop were pooled together and loaded onto a HiLoad 16/600 Superdex^TM 200 pg column equilibrated with buffer C (10 mM Tris-HCl, pH 8, 300 mM NaCl containing 5% glycerol, 0.2 mM TCEP). Eluted fractions were analysed using SDS-PAGE to determine the purity and homogeneity of the PfHop protein. Authenticity of the purified protein was confirmed by sequencing using MALDI-TOF mass spectrometry at the Biocenter Oulu Proteomics Core Facility, Oulu University, Finland. The protein concentration was determined by measuring the UV absorbance at 280 nm using a Nanodrop ND100 (ThermoFisher Scientific, USA).

The molecular weight of PfHop was determined using multi-angle static light scattering (MALS) coupled to size exclusion chromatography using a Superdex S200 Increase 10/300 GL column (GE Healthcare). The column, equilibrated with buffer C, was coupled to a mini DAWN TREOS MALS detector (Wyatt Technology, Germany) and an ERC RefraMAx520 differential refractometer (ERC, Germany). 100 μg of PfHop in buffer C was injected into the column using a flow rate of 0.5 ml/min. BSA and ovalbumin were used as molecular-weight controls. The molecular weight of PfHop was determined based on the measured light scattering at three different angles and the refractive index using the ASTRA software version 6.1.5.22 (Wyatt Technology, Germany).

2.3 Investigation of the secondary structure of PfHop

The secondary structure of PfHop was investigated using synchrotron radiation (SR) and conventional circular dichroism (CD) spectroscopy. The spectral measurements were conducted at the UV-CD12 beam line (Anka, Karlsruhe) under temperature-controlled conditions. PfHop at a concentration of 0.5 mg/ml dialysed in buffer D (10 mM K₃PO₄, pH 7.0, 150 NaF) was analysed using a 98.56 μm path length round cell cuvette (Suprasil, Hellma Analytics, Germany) at a constant temperature of 10°C. A total of 3 full spectral scans were recorded from 280 to 175 nm and averaged. CD spectroscopy experiments were done using a Jasco J-1500 CD spectrometer (JASCO, Tokyo, Japan) and Chirascan CD Spectrometer (Applied Photophysics, UK) with a temperature-controlled Peltier. Recombinant proteins at a final concentration of 2 μM were analysed using a 2-mm path-length quartz cuvette (Hellma). Spectral scans were recorded from 250 to 180 nm and averaged for least 3 scans. The SRCD data were processed and deconvoluted using the Dichroweb server [29] and the CONTINLL algorithm with the SP175 reference set [30]. To further predict the secondary structure content of PfHop, the BeStSel server (http://bestsel.elte.hu/; [31, 32]) and the Phyre2 server (http://sbg.bio.ic.ac.uk/phyre2/; [33]) were also used. In order to investigate the heat stability of PfHop, the protein was subjected to increasing temperature (10°C to 90°C using 5°C intervals for SRCD and 20 to 90°C for CD) and full spectra recorded when the temperature was raised from 20°C—90°C. Similarly, spectra were recorded upon subjecting the protein to a downward shift in temperature from 90°C to 20°C. Melting curves were plotted by monitoring the CD signal at 192, 210 and 220 nm using the Jasco J-1500 CD spectrometer and further validated using Chirascan CD spectrometer (Applied Photophysics, UK). The CD spectral measurements were expressed as ratio of signal recorded at a particular temperature compared to the SRCD signal recorded at 10°C. This facilitated estimation of the folded protein fraction of the protein at the respective temperature as previously described [34, 35]. The tertiary structure of the protein was probed in the presence of varying concentrations of denaturants, urea (0–8 M) and guanidine hydrochloride (0–6 M).

Fluorescence spectra were recorded following initial excitation at 295 nm and emission was determined at wavelength range of 300 nm to 400 nm using JASCO FP-6300 spectrofluorometer (JASCO, Tokyo, Japan).

2.4 Small-angle X-ray scattering analysis for PfHop shape determination

Synchrotron small-angle X-ray scattering (SAXS) data were collected on the EMBL Hamburg Outstation beam line P12 at PETRA III/DESY (Hamburg). PfHop (2.2 mg/ml) and buffer samples were exposed to X-rays with a wavelength of 1.240 Å for 0.045 s. Pre-processed data were further analysed with the ATSAS software package [36]. The distance distribution calculation and ab initio modelling were performed using GNOM [37] and the GASBOR package [38], respectively. Human Hop TPR1 (1ELW; [39]) and bakers’s yeast TPR2AB domains (3QU3; [23]) were manually fitted in the ab initio envelope using PyMOL 2.3.2 (Schrödinger, USA). An R_g value of 5.3 nm was determined visually from the linear part of the low scattering angles (0.0087–0.0715 nm^-1) using PRIMUS [40]. The D_max for PfHop was estimated as 24 nm, also using PRIMUS.

3. Results

3.1 Oligomeric state of recombinant PfHop

Recombinant PfHop was purified using nickel affinity chromatography as previously described [18]. The protein was further purified using ion exchange and subsequently subjected to size exclusion chromatography (S1 Fig).

Hop has been reported to exist as either monomer [21, 41, 42] or dimer [43, 44], andas largely monomeric, forming weak dimers [45]. Based on size exclusion chromatography, PfHop eluted as an elongated monomer under reducing conditions. A small fraction of dimer, likely due to partial oxidation, could be seen in some batches (S1C Fig, lanes 1–5). A recent study [46] reported that PfHop exists as a dimer. However, using multi-angle light scattering coupled to size exclusion chromatography we observed that PfHop occurs as a monomer of 73 kDa (Fig 1). This is 8% larger than the calculated theoretical molecular weight 67.6 kDa. A previous independent study [20] similarly reported an apparently higher molecular weight for STI1 that was determined using gel filtration. As a control, we determined molecular weights for bovine serum albumin (73 kDa) and ovalbumin (45 kDa), which were 10% and 6%, respectively, larger than their calculated theoretical molecular weights.

Fig 1 — Size exclusion chromatography of PfHop displays a single peak. The molecular weight of the peak calculated using static light scattering, shown as a black line, represents a PfHop monomer.

3.2 Analysis of the secondary structure of PfHop

To confirm the folding state and secondary structure composition of recombinant PfHop, synchrotron radiation circular dichroism (SRCD) spectroscopy was conducted. SRCD spectra were recorded between 175 and 280 nm at 10°C. The PfHop spectra exhibited 2 negative minima around 222 and 208 nm and a positive peak at 194 nm (Fig 2A), characteristic of a predominantly α-helical protein [47] as previously reported [46]. Deconvolution of the spectra with Dichroweb indicated a predominantly α-helical structure comprising 77% α-helices (Table 1). This was supported by predictions from BeStSel and Phyre2 (S1 Table). The helical content estimated here for PfHop is comparable to that for Leishmania braziliensis Hop (LbHop) which was reported to be around 75% [42]. Notably, the PfHop helical content of 77% we observed here is much higher than that the 57% obtained for the same protein in a recent independent study [46]. While, it is not clear why such a wide discrepancy has been reported for PfHop helical content, our findings reconcile with that reported for its close homolog, LbHop [42]. Overall, the predominantly α-helical structure of PfHop is consistent with the predicted three-dimensional model of PfHop which showed that all its three TPR motifs are α-helical in nature [18]. Furthermore, based on the previously generated three-dimensional model of PfHop, residues of PfHop that are implicated in making direct contact with PfHsp70-1/PfHsp90 are positioned within the grooves of the α-helical TPR domains [18].

Fig 2 — (A) SRCD spectrum of full-length PfHop. SRCD spectral scans monitoring denaturation of PfHop upon exposure to increasing heat stress (10°C to 90°C). (B) Shown is the CD spectrum of PfHop monitored at 222 nm upon thermal denaturation by upscaling temperature from 20°C to 90°C. Similarly, the CD spectrum for the renaturation attempt of PfHop upon temperature downscale from 90°C to 20°C is illustrated. The thermal transitions (Tm₁ and Tm₂) are shown. (C) The folded fraction of PfHop as a function of temperature was monitored using CD signals at 192, 210 and 220 nm. (D) Urea-induced unfolding of PfHop is shown. (E) Represents the fluorescence emission spectra of PfHop monitored at 300–450 nm after an initial excitation at 295 nm. The recombinant PfHop protein tryptophan fluorescence emission spectra were recorded under various GdHCl and urea concentrations. Notable, is the red spectral shift obtained for of PfHop exposed to various GdHCl and urea concentrations.

Table 1. SAXS parameters for PfHop.

Sample	Rg (nm)	Dmax (Å)	MW (kDa)	Expected MW (kDa)
SLS			73	67.6
SAXS	5.3	240		67.6

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PfHop mediates interaction between PfHsp70-1 and PfHsp90 [18, 20], and its expression is heat-induced [18]. The roles of these two chaperones become particularly important when the parasite is subjected to physiological stress, such as during clinical malaria fever episodes [48]. It is therefore important that heat shock proteins of parasite origin exhibit resilience to heat stress conditions. PfHsp70-1 is stable at high temperatures and is most active at 48 ºC—50 ºC and is known to retain its ATPase activity at up to 80 ºC [6; 34]. However, it remains to be established whether PfHop exhibits the same resilience to heat stress. To probe this, we investigated the heat stability of recombinant PfHop in vitro. As a control, the denaturation of PfHop exposed to urea (0–8 M) was also monitored using CD (Fig 2C). Next, we monitored the folded fraction of PfHop in response to exposure to increased temperature conditions using SRCD (10°C to 90°C) (Fig 2A) and CD spectrometry (assay was conducted at 20°C—90°C; Fig 2B). PfHop appeared stable at temperatures lower than 40°C. However, at higher temperatures the protein lost its fold, and only 50% of the protein retained its folded state at 45°C (Fig 2C). Notably, the spectra suggest that the protein simultaneously loses both its α-helical and β-compositions in response to heat stress through two unfolding transitions (Tm₁ and Tm₂) (Fig 2B). CD spectrometry was used to monitor heat-induced denaturation of PfHop at a temperature range of 20°C—90°C (Fig 2B). The double phased unfolding transitions we observed here closely mirror those reported for PfHop in an independent study [46]. An attempt to refold the heat-denatured PfHop by lowering temperature from 90°C—20°C showed that the protein subjected to temperatures above 70°C could not refold. Our findings suggest that PfHop is less stable to heat stress than PfHsp70-1, whose ATPase activity was found to be optimal around 50°C [34]. In addition, PfHsp70-1 exhibits chaperone activity (suppressing heat induced aggregation of protein) at 48°C [6].

Furthermore, tryptophan fluorescence spectroscopy was conducted to monitor the tertiary structural organisation of PfHop in the presence of varying amounts of urea and guanidine hydrochloride (GdHCl). A red shift was observed with maximum peaks at 350 nm (associated with 6 M GdHCl) and 343 nm (associated with 8 M urea) (Fig 2E). PfHop was more sensitive to GdHCl, which is a stronger denaturant. This is in agreement to a previous observation for PfHsp70-1 protein [34].

3.3 Low-resolution structure of PfHop in solution

In order to gain further insight into the structure of PfHop, we determined its low-resolution structure in solution using SAXS (Fig 3). The X-ray scattering curve (Fig 3A), the Kratky plot (Fig 3B), and the distance distribution function (Fig 3C) together indicate that PfHop is an elongated protein with a maximum dimension of approximately 24 nm (Table 1). PfHop consists mostly of folded parts connected by flexible linkers. Thus, as expected, the TPR domains likely are arranged like pearls on a string. The distance distribution function (Fig 3C) indicates at least two stable domains with maxima at ~3 and ~5 nm within the D_max of 240 Å. The D_max obtained for PfHop falls within the range obtained for other Hop homologues; 180 Å for LbHop [42], 193 Å for Hop [43], 230 Å for PfHop [46] and 260 Å for STI1 [20]. Altogether, these values confirm Hop to generally assume an extended conformation across species. An ab initio dummy residue model calculated using GASBOR (Fig 3D) is consistent with the above data and shows an excellent fit to the experimental data (Fig 3) with a χ² value of 1.06. The model consists of an elongated shape with some more compact regions, as observed for STI1 [20]. This model visually fits perfectly to crystal structures of human and yeast Hop TPR1 (1ELW, [39]) as well as that of the TPR2AB (3UQ3, [23]) domain (Fig 3D).

4. Discussion

PfHop is thought to facilitate the functional cooperation between PfHsp70-1 and PfHsp90, both prominent cytosolic molecular chaperones of P. falciparum [18, 19]. The Hsp70-Hop-Hsp90 pathway plays an important role in cellular development, as it facilitates folding and maturation of proteins, such as kinases and steroid hormone receptors [49]. Inhibition of both PfHsp90 and PfHsp70-1 leads to parasite death [7, 24, 26], making these molecular chaperones potential antimalarial drug targets. In the current study, we demonstrated that PfHop is unstable at temperatures above 40°C. Furthermore, the protein is denatured at elevated temperatures through two distinct unfolding transitions (Fig 2), in agreement with a recent independent study [46]. The two unfolding transitions are consistent with a protein possessing domains of varied conformational stability. This is consistent with the multi-domain nature of PfHop as observed in the current study. In agreement with this, the N-terminus of Hop is reportedly flexible while its C-terminus is deemed to be compact [20].

We previously observed that PfHop, PfHsp70-1, and PfHsp90 occur in a complex, and that PfHop directly associates with the EEVD domains of PfHsp70-1 and PfHsp90 [18, 19]. This suggests that PfHop modulates functional cooperation between PfHsp70-1 and PfHsp90. The development of clinical malaria is associated with body temperature rising to 41.6°C. At such high temperatures, the role of heat shock proteins in maintaining proteostasis becomes unquestionably important, as is evidenced by the elevated expression of key molecular chaperones, such as PfHsp70-1 and PfHsp90 [7, 50]. It is intriguing to imagine how PfHsp70-1 and PfHsp90 cooperate in the presence of limiting levels of PfHop, as occurs during sustained heat stress conditions induced by malaria fever. It is possible that under extended stress conditions, the role of Hop becomes less vital and that perhaps Hsp70 and Hsp90 may directly interact. Indeed, a study showed that despite lack of Hop in E. coli, Hsp70 and Hsp90 from E. coli are capable of direct interaction [15]. In addition, Hsp70 and Hsp90 chaperones resident in yeast cytosol and mouse ER were reported to directly bind without Hop as an adaptor [13, 14]. In a previous study, we observed complexes of PfHsp70-1 and PfHsp90 in which PfHop was present based on size exclusion chromatography of parasite lysates [18]. However, we also observed eluates representing a complex of PfHsp70-1 and PfHsp90, in which PfHop was absent [18]. A non-canonical Hop homologue from Caenorhabditis elegans lacks the TPR1 domain, hence it is thought to be biased towards binding to Hsp90 than Hsp70 [51]. Altogether, our findings and those of others suggest that the function of Hop may vary across species and may also depend on the prevailing cellular physiological conditions.

Findings on the oligomeric status of Hop have remained controversial as independent studies have reported it to be either monomeric [41] or dimeric [44], or largely monomeric but forming also weak dimers [45]. In the current study, we sought to establish the oligomeric status of PfHop. Based on size-exclusion chromatography and multi-angle static light scattering, we conclude that PfHop occurs as a monodisperse monomer (Fig 1). This conflicts with a recent study which reported PfHop to exist as a dimer [46]. It is important to note that we also observed a dimeric fraction which we think is on account of unspecific disulphide bridge formation that occurs under non-reducing conditions. Interestingly, our previous findings using SPR showed that PfHop self-associates with higher affinities [19], suggesting that PfHop may form oligomers. It is possible that PfHop self-association occurs transiently, hence may be detected by techniques such as SPR but not by other techniques such as SEC. This could account for the conflicting findings. On the other hand, human Hop has been reported to form dimers [44, 52, 53], but it remains to be confirmed whether these are of functional significance. In addition, Hop has also been suggested to form elongated monomers, which are difficult to resolve using gel filtration [41]. Indeed, the low-resolution solution structure determined by SAXS shows that PfHop is a highly elongated and multidomain protein. This agrees with several previous independent studies [20, 42, 43, 46]. Notably, we observed that the folded domains of PfHop are organised like beads on a string. This is consistent with the predicted concave nature of its predominantly α-helical TPR motifs [18]. TPR motifs of human Hop have been described to occur as grooves into which the C-terminal EEVD motifs of Hsp90 and Hsp70 bind in extended form [39]. Our PfHop SAXS data is consistent with this proposed model.

Altogether, our findings established that PfHop is an elongated, predominantly α-helical, monomeric, protein. Its heat stability is lower than that reported for PfHsp70-1, suggesting that its function may be compromised at high temperatures associated with clinical malaria progression.

Supporting information

S1 Fig. Purification of PfHop by ion exchange and size exclusion chromatography.

(A) SDS-PAGE analysis of the five samples collected from the main peak (lanes 1–5). Ion exchange chromatography of PfHop purification was monitored at 280 nm. Protein that bound to the column was then eluted under NaCl gradient. The five fractions obtained were pooled together for subsequent SEC analysis. (B) SDS-PAGE analysis of several fractions (lanes 1–11) of PfHop obtained by SEC are shown. (C) SDS-PAGE analysis of several fractions (lanes 1–12) of PfHop obtained by SEC are shown.

(DOCX)

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S2 Fig. Mass spectrometry sequencing data for PfHop.

(DOCX)

Click here for additional data file.^{(531.3KB, docx)}

S1 Table. PfHop secondary structure content.

(DOCX)

Click here for additional data file.^{(16.4KB, docx)}

S1 Raw Images

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Click here for additional data file.^{(69.9KB, pdf)}

Acknowledgments

We thank Drs. Arne Raasakka, Erik Hallin, and Juha Kallio for SRCD and SAXS data collection as well as help with data processing. We acknowledge the KIT light source for provision of instruments at the beamline UV-CD12 of the Institute of Biological Interfaces (IBG2), and we would like to thank the Institute for Beam Physics and technology (IBPT) for the operation and storage ring, the Karlsruhe Research Accelerator (KARA). We would like to acknowledge the Biophysics, Structural Biology, and Screening (BiSS) facilities at University of Bergen for access to the static light scattering instrument. We are grateful to the office of University of Stellenbosch Central Analytical Facilities for allowing us access to use their Chirascan CD Spectrometer.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

1. IK, Sigrid Jusélius Foundation; https://sigridjuselius.fi/en/ 2. AS, L1/402/14–1; Deutsche Forchungsgemeinshaft; https://www.validate-network.org/event/german-african-cooperation-projects-in-infectiology-dfg 3. AS; 75464; 92598; National Research Foundation/Department of Science & Innovation of South Africa; https://www.nrf.ac.za/ 3. TZ; 111989; National Research Foundation of South Africa; www.nrf.ac.za

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PLoS One. doi: 10.1371/journal.pone.0226657.r001

Decision Letter 0

Didier Picard

9 Jan 2020

PONE-D-19-33340

Biophysical analysis of Plasmodium falciparum Hsp70-Hsp90 organizing protein (PfHop) reveals a monomer that is characterised by folded segments connected by flexible linkers

PLOS ONE

Dear Prof. Shonhai,

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Moreover, previous evidence from the same lab indicating the existence of dimers should not be shrugged of as lightly as it is done in the Discussion on page 12 ("However, a dimeric fraction is observed in non-reducing conditions, indicating unspecific disulphide bridge formation, which would explain some of the previous data [12]"). This needs to be more thoroughly addressed (if necssary also experimentally).

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Reviewer #1: This manuscript from Makumire et al. examines the biophysical properties of the Hsp70/Hsp90 co-chaperone Hop from P. falciparum (PfHop). The main conclusions are:

1) PfHop is primarily monomeric

2) PfHop has folded segments connected by flexible linkers

3) PfHop is unstable at temperatures over 40C

The third conclusion is important because is suggests that PfHop may lose part of its structure/function under fever conditions associated with clinical malaria.

Most of the conclusions are well-supported although I think one control experiment is required. The authors need to establish whether the thermal denaturation is reversible or irreversible. Specifically, an experiment is needed like that shown in Fig2b where the temperature is first increased up to 80C and then decreased back down to 20C. If the denaturation is reversible then these curves should overlap. If it is not reversible then the thermal denaturation results should be interpreted cautiously. I think this is an essential control.

Minor points:

Fig 2A: CD for protein samples is usually reported as molar ellipticity (theta) with units of deg*cm^2/dmol.

Were replicate experiments performed for the thermal melt? If no, then the authors should confirm the reproducibility. Some data on reproducibility should be shown in the text or figures.

The folded fraction (y-axes) in Figures 2B&C is not well-defined because it is not clear whether 80C represent the fully denatured baseline. Also, because it is not whether the folding is cooperative or multi-state it is not clear how to convert the CD signal into a fraction folded value. I would advise plotting the y-axes as the raw CD signal.

SAXS analysis of Hop has been performed in other studies in addition to the Rohl 2015 study (for example Onuoha et al. JMB 2008). It may be helpful to briefly discuss how your SAXS results compares with previous studies.

The discussion cites literature showing Hsp90/Hsp70 directly interacting in the absence of Hop. The ER-specific Hsp70/Hsp90 (BiP/Grp94) also show a direct interaction (see Sun et al. JBC 2019).

Best Regards,

Timothy Street

Reviewer #2: In the manuscript, Makumire and colleagues use a number of biophysical techniques to analyse the Plasmodium falciparum orthologue of the Hsp70/Hsp90 co-chaperone, Hop. The authors produced the protein recombinantly in E.coli as a his-tag fusion and purified it using IMAC and ion exchange chromatography. The purified protein subsequently underwent partial biophysical characterisation using CD/SRCD, SAXS and tryptophan fluorescence. They conclude that PfHop is an elongated monomer comprised mostly of alpha helical secondary structure. In addition, they conclude that PfHop is unstable at temperatures above 40 °C, while its partner protein PfHsp70-1 is stable at much higher temperatures.

Overall the manuscript is straightforward and the data presented are consistent with previous reports, and perhaps not surprising given the sequence similarity and molecular modelling of the PfHop structure, as well as our understanding of the structural and biophysical features of Hop from other organisms. This study provides experimental data to support bioinformatics models and demonstrate that structurally PfHop is similar to Hop from other organisms. A range of appropriate biophysical methods are used in the study, and the experiments appear to have been performed appropriately. Perhaps one limitation is that all of the analyses were conducted with a single concentration of PfHop.

This study is very similar to another one from the Borges lab (doi: 10.1016/j.bbapap.2019.140282.) which has just been published. In this recent paper, the group describes the biophysical characterisation of PfHop, including a number of similar or identical methods to this study (SAXS, tryptophan fluorescence, thermal unfolding and AUC). The fact that this recent paper is so similar should not prevent publication of the current study, but it does provide a good comparison for data generated in two independent labs. The PfHop study is very recent and hence it is possible that this paper may not have been identified prior to submission which is why it is not included. Nevertheless, this study should be an essential comparison to the current manuscript.

The authors have been measured in their interpretation, and in my opinion, could perhaps improve the depth of comparison of their data to other key reports on biophysical characterisation of Hop orthologues without over-stating conclusions. The recent reports on direct interactions between Hsp70 and Hsp90 in eukaryotes needs to be given more priority and included in the introduction, not only the discussion. These studies redefine our understanding of the role that Hop plays in the Hsp70-Hsp90 complex. It is clear Hop is no longer essential for interaction in eukaryotes, although this does not mean it is not required under specific conditions. Therefore, it is not necessarily completely unexpected or surprising that PfHsp70 and PfHsp90 may be able to directly interact in the absence of PfHop.

PfHop is proposed to be instable at temperatures above 40 °C, which has implications for the PfHsp70-PfHsp90 chaperone complex during fever in malaria (where temperatures can reach this temperature). However, the conclusion of instability above 40 °C on the data presented is subjective at best. The authors should calculate the Tm which would give a more direct and comparable temperature stability. In addition, the current discussion fails to explicitly compare this with that reported for human Hop (in the region of 52 degrees C) as indicated by the study from the Regan group. This is important since the implication is that human Hop may be more stable than PfHop, which could imply biochemical differences between the two chaperone systems despite high sequence and structural similarity. In addition, the stability studies have been conducted in vitro and one needs to be cautious in directly extrapolating findings to an in vivo scenario where the protein may behave differently due to the environment. While I think the conclusion can be made, this aspect is not highlighted at all in the manuscript.

What is the relevance of the data shown in Fig 2D? How this relates to the rest of the analysis was not clearly articulated to me.

It may be better to include Fig 3D as its own figure so that it can be made larger.

The SAXS results need to be compared to the studies from the Jackson lab (Onuoha etal J Mol Biol. 2008 Jun 13; 379(4):732-44), who did SAXS on human Hop-Hsp90 complexes and is currently not referenced in the manuscript, and from the Borges lab who have done this (and other biophysical characterisation) on both LmHop (doi: 10.1016/j.abb.2016.04.008.) and PfHop.

How confident are the authors of the SEC-MAL data which appeared to consistently over-estimate the molecular weight of proteins studied? There are other studies on BSA at least which give a molecular weight at 64 kDa?

There are some minor missing articles and inconsistencies e.g. Tpr vs TPR in the manuscript that can be easily corrected with appropriate proofreading.

A minor final point, the abstract makes reference to ‘select number of cellular proteins required for cyto-protection……’. To the best of my knowledge (and I could of course be wrong), there has been limited characterisation of bona fide chaperone clients in malaria and hence, while this statement is likely true, has it been experimentally verified?

The authors are encouraged to deposit the SAXS data in the SASBDB and link the accession number to the manuscript.

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Reviewer #1: Yes: Timothy Street

Reviewer #2: No

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PLoS One. 2020 Apr 28;15(4):e0226657. doi: 10.1371/journal.pone.0226657.r002

Author response to Decision Letter 0

27 Mar 2020

Reviewer #1: This manuscript from Makumire et al. examines the biophysical properties of the Hsp70/Hsp90 co-chaperone Hop from P. falciparum (PfHop). The main conclusions are:

1) PfHop is primarily monomeric

2) PfHop has folded segments connected by flexible linkers

3) PfHop is unstable at temperatures over 40C

The third conclusion is important because is suggests that PfHop may lose part of its structure/function under fever conditions associated with clinical malaria.

Authors Response:

We appreciate the reviewer’s comment and we have repeated the thermal denaturation and included the data for renaturation in Fig 2b as recommended. Indeed, there is no overlap between the two curves suggesting that exposure of the PfHop to temperatures above 70°C irreversibly denatures the protein.

Minor points:

Reviewer: Fig 2A: CD for protein samples is usually reported as molar ellipticity (theta) with units of deg*cm^2/dmol.

Author’s Response: SRCD measurements were previously presented in delta epsilon and are now converted and reported as such in molar ellipticity [(θ)m]. Using the formula Δε = (θ)m * Nr / 3298 (where, Delta Epsilon = Δε; (θ)m = Molar Ellipticity (θ)m; and Nr represents the number of amino acids in the protein = 564 :

Reviewer: Were replicate experiments performed for the thermal melt? If no, then the authors should confirm the reproducibility. Some data on reproducibility should be shown in the text or figures.

Authors Response: Thermal denaturation was conducted using Synchrotron radiation circular dichroism spectroscopy. A repeat of the thermal denaturation was conducted, however using conventional CD Jasco CD and Chirascan Cd spectrometers and a similar denaturation patterns were observed. The thermal melts results from two different sets of equipment and experimental set up with 3 scans averaged showing that the thermal melt is reproducible.

Reviewer: The folded fraction (y-axes) in Figures 2B&C is not well-defined because it is not clear whether 800C represent the fully denatured baseline. Also, because it is not whether the folding is cooperative or multi-state it is not clear how to convert the CD signal into a fraction folded value. I would advise plotting the y-axes as the raw CD signal.

Authors Response: The folded fraction represents the proportion of properly folded protein at a given temperature or concentration of denaturant. The following statement from the Methods section clarifies how this assay was conducted, “The CD spectral measurements were expressed as ratio of signal recorded at a particular temperature compared to the SRCD signal recorded at 10 °C. This facilitated estimation of the folded protein fraction of the protein at the respective temperature using as previously described protocols [34; 35].” The cited reference papers have details on the functions that were used to calculate the folded fraction as follows;

The following statements were included in the methods to clarify the conversion to folded fraction, “The folded state of the proteins at any given temperature was determined as follows (Misra and Ramachandran, 2009):

([θ]t – [θ]h) / ([θ]l – [θ]h) Equation 1

Where (θ)t is the molar ellipticity at any given temperature, (θ)h at highest temperature, and (θ)l at lowest temperature, respectively.”

“The folded state of the proteins at each urea concentration, the following equation (Eq. 1) modified to:

([θ]u - [θ]h) / ([θ]l - [θ]h)

Where [θ]u is the molar ellipticity at any given urea concentration, [θ]h at highest urea concentration, and [θ]l at lowest urea concentration, respectively.”

Reviewer: SAXS analysis of Hop has been performed in other studies in addition to the Rohl 2015 study (for example Onuoha et al. JMB 2008). It may be helpful to briefly discuss how your SAXS results compares with previous studies.

Authors Response: The SAXS results for PfHop have been compared with those of other Hop homologues and also discussed accordingly. The following statements were included, “In order to gain further insight into the structure of PfHop, we determined its low-resolution structure in solution using SAXS (Figure 3). The X-ray scattering curve (Figure 3A), the Kratky plot (Figure 3B), and the distance distribution function (Figure 3C) together indicate that PfHop is an elongated protein with a maximum dimension of approximately 24 nm (Table 1). PfHop consists mostly of folded parts connected by flexible linkers. Thus, as expected, the TPR domains likely are arranged like pearls on a string. The distance distribution function (Figure 3C) indicates at least two stable domains with maxima at ~3 and ~5 nm within the Dmax of 240 Å . The Dmax obtained for PfHop falls within the range obtained for other Hop homologues; 180 Å for LbHop [42], 193 Å for Hop [43], 230 Å for PfHop [46] and 260 Å for STI1 [20]. Altogether, these values confirm Hop to generally assume an extended conformation across species. An ab initio dummy residue model calculated using GASBOR (Figure 3D) is consistent with the above data and shows an excellent fit to the experimental data (Figure 3) with a χ2 value of 1.06. The model consists of an elongated shape with some more compact regions, as observed for STI1 [20]. This model visually fits perfectly to crystal structures of human and yeast Hop TPR1 (1ELW, [39]) as well as that of the TPR2AB (3UQ3, [23]) domain (Figure 3D).”

“Indeed, the low-resolution solution structure determined by SAXS shows that PfHop is a highly elongated and multidomain protein. This agrees with several previous independent studies [20; 42; 43; 46]. Notably, we observed that the folded domains of PfHop are organised like beads on a string. This is consistent with the predicted concave nature of its predominantly α-helical TPR motifs [18]. TPR motifs of human Hop have been described to occur as grooves into which the C-terminal EEVD motifs of Hsp90 and Hsp70 bind in extended form [39]. Our PfHop SAXS data is consistent with this proposed model.”

Reviewer: The discussion cites literature showing Hsp90/Hsp70 directly interacting in the absence of Hop. The ER-specific Hsp70/Hsp90 (BiP/Grp94) also show a direct interaction (see Sun et al. JBC 2019).

Authors Response: The following statement was added to the discussion citing recent literature, “Interestingly, Hsp70 and Hsp90 resident in the cytosols of E. coli and yeast as well as their homologs localized to mouse ER were shown to interact directly independent of Hop mediation [11; 12; 13; 14].” Reference 14 represents work by Sun et al., 2019 which we now included.

Authors Responses: As this is a follow-up study using optimized protocols such as work from Gitau et al.,2012 [18]; Zininga et al., 2015 [19]; among other studies on the same/related proteins, the current protocol was thus guided by guidelines from such work. However, for distinct assays, we used protein concentrations that were appropriate for each assay as outlined in the methods section. Below we highlight extracts of some of the assays, highlighting protein concentrations used:

1. SRCD assays: “PfHop at a concentration of 0.5 mg/ml dialysed in buffer D (10 mM K3PO4, pH 7.0, 150 NaF) was analysed using a 98.56 µm path length round cell cuvette (Suprasil, Hellma Analytics, Germany) at a constant temperature of 10°C.”

2. SEC/SLS: 100 µg of PfHop in buffer C was injected into the column using a flow rate of 0.5 ml/min. BSA and ovalbumin were used as molecular-weight controls. The molecular weight of PfHop was determined based on the measured light scattering at three different angles and the refractive index using the ASTRA software version 6.1.5.22 (Wyatt Technology, Germany).

3. SAXS analyses: PfHop (2.2 mg/ml) and buffer samples were exposed to X-rays with a wavelength of 1.240 Å for 0.045 s. Pre-processed data were further analysed with the ATSAS software package [27].

Reviewer: This study is very similar to another one from the Borges lab (doi: 10.1016/j.bbapap.2019.140282.) which has just been published. In this recent paper, the group describes the biophysical characterisation of PfHop, including a number of similar or identical methods to this study (SAXS, tryptophan fluorescence, thermal unfolding and AUC). The fact that this recent paper is so similar should not prevent publication of the current study, but it does provide a good comparison for data generated in two independent labs. The PfHop study is very recent and hence it is possible that this paper may not have been identified prior to submission which is why it is not included. Nevertheless, this study should be an essential comparison to the current manuscript.

Authors Response: The findings from this study have now been compared to the recent PfHop paper (Silva et al., 2019 [46]) and discussions made accordingly in the various sections. Changes have been tracked.

Reviewer: The authors have been measured in their interpretation, and in my opinion, could perhaps improve the depth of comparison of their data to other key reports on biophysical characterisation of Hop orthologues without over-stating conclusions. The recent reports on direct interactions between Hsp70 and Hsp90 in eukaryotes needs to be given more priority and included in the introduction, not only the discussion. These studies redefine our understanding of the role that Hop plays in the Hsp70-Hsp90 complex. It is clear Hop is no longer essential for interaction in eukaryotes, although this does not mean it is not required under specific conditions. Therefore, it is not necessarily completely unexpected or surprising that PfHsp70 and PfHsp90 may be able to directly interact in the absence of PfHop.

Authors Responses: The data has been compared to other previous studies characterizing various Hop homologues. The direct interaction of Hsp70/Hsp90 was revisited and aspects included in the introduction. The introduction now reads as, “Stress inducible protein 1 (STI1) was first described in mouse [10], and now also known as Hsp70-Hsp90 organizing protein (Hop), acts as a module that allows Hsp70 and Hsp90 to interact stably, thereby facilitating substrate transfer from Hsp70 to Hsp90. Interestingly, there are recent findings that redefine our understanding of the role that Hop plays in the Hsp70-Hsp90 complex. Several studies have reported the direct interactions between Hsp90 and Hsp70 (Nakamoto et al., 2014; Kravats et al., 2017; 2018, Sun et al., 2019). Using Hsp90 and Hsp70 from bacteria and yeast, direct Hsp90/Hsp70 interaction was reported to occur via a region in the middle domain of Hsp90 (Nakamoto et al., 2014; Kravats et al., 2017; 2018). The ER Hsp70/Hsp90 (BiP/Grp94) were also shown to interact directly in a nucleotide dependent manner (Sun et al., 2019). Given that there is no Hop homolog in E. coli, a direct association between Hsp90 and Hsp70 in E. coli is important (Genest et al., 2016). The Hop gene is not essential in yeast (Reidy et al., 2018), Trypanosoma cruzi and Trypanosoma brucei although in the absence of Hop certain functions are affected (Schmidt et al., 2018). Hop may not be essential in some organisms but may be required under certain conditions. This might explain the need for Hsp90/Hsp70 direct interaction (Nakamoto et al., 2014). It remains to be investigated whether this direct interaction is conserved among species. Hop has been proposed as a potential drug target (Gitau et al., 2012; Zininga et al., 2015b). However, this would only apply in cases where Hop is essential for example in cancer (Röhl et al., 2014) and Leishmaniasis (Morales et al., 2010; Hombach et al., 2013).

Hop is a conserved and stress inducible protein that possesses three tetratricopeptide repeats (TPR): TPR1, TPR2A and TPR2B [10]. Both Hsp70 and Hsp90 interact with Hop via the C-terminal EEVD motif, present in the two molecular chaperones [1119; 23; 12]. Hop interacts with Hsp70 and Hsp90 via its TPR1 and TPR2A domains, respectively [1123]. While for a long time the role of the TPR2B domain of Hop has remained largely elusive, it is now thought that Hsp70 first binds to the TPR1 domain of Hop before switching to the TPR2B domain to facilitate substrate transfer to Hsp90 [20; 23].’’

Reviewer: PfHop is proposed to be instable at temperatures above 40 °C, which has implications for the PfHsp70-PfHsp90 chaperone complex during fever in malaria (where temperatures can reach this temperature). However, the conclusion of instability above 40 °C on the data presented is subjective at best. The authors should calculate the Tm which would give a more direct and comparable temperature stability. In addition, the current discussion fails to explicitly compare this with that reported for human Hop (in the region of 52 degrees C) as indicated by the study from the Regan group. This is important since the implication is that human Hop may be more stable than PfHop, which could imply biochemical differences between the two chaperone systems despite high sequence and structural similarity. In addition, the stability studies have been conducted in vitro and one needs to be cautious in directly extrapolating findings to an in vivo scenario where the protein may behave differently due to the environment. While I think the conclusion can be made, this aspect is not highlighted at all in the manuscript.

Authors Response: The PfHop Tm is the temperature at which 50 % of the protein is folded. We already have a folded fraction curve (Figure 2B shown as the temperature transitions that the protein undergoes during thermal unfolding. The calculated Tm for the protein is 50.8 °C and we prefer going with the thermal transitions of the protein unfolding.

Reviewer: What is the relevance of the data shown in Fig 2D? (now 2E) How this relates to the rest of the analysis was not clearly articulated to me.

Authors Response: Figure 2D shows the tertiary structure chemical perturbation of PfHop in the presence of denaturants (urea and guanidine chloride). This was monitored by tryptophan fluorescence to mirror the chemical perturbations observed in secondary structure analysis using CD spectra in Figure 2D.

Reviewer: It may be better to include Fig 3D as its own figure so that it can be made larger.

The SAXS results need to be compared to the studies from the Jackson lab (Onuoha et al J Mol Biol. 2008 Jun 13; 379(4):732-44), who did SAXS on human Hop-Hsp90 complexes and is currently not referenced in the manuscript, and from the Borges lab who have done this (and other biophysical characterisation) on both LmHop (doi: 10.1016/j.abb.2016.04.008.) and PfHop.

Authors Response: The SAXS data for PfHop has been compared with other SAXS studies on various Hop homologs as suggested.

Reviewer: How confident are the authors of the SEC-MAL data which appeared to consistently over-estimate the molecular weight of proteins studied? There are other studies on BSA at least which give a molecular weight at 64 kDa?

Authors Response: We are conscious of the limitations of the SEC-MAL data and do discuss this in our MS.

Reviewer: There are some minor missing articles and inconsistencies e.g. Tpr vs TPR in the manuscript that can be easily corrected with appropriate proofreading.

Authors Response: Inconsistencies were identified and rectified as in, “Human Hop TPR1 (1ELW; 0) and bakers’s yeast TPR2AB domains (3QU3; 11) were manually fitted in the ab initio envelope using PyMOL 2.3.2 (Schrödinger, USA). Any other minor inconsistencies were corrected in the text and highlighted through the track changes option.

Reviewer: A minor final point, the abstract makes reference to ‘select number of cellular proteins required for cyto-protection……’. To the best of my knowledge (and I could of course be wrong), there has been limited characterisation of bona fide chaperone clients in malaria and hence, while this statement is likely true, has it been experimentally verified?

Authors Response: The statement was reworded to “The cooperation of heat shock protein (Hsp) 70 and 90 is thought to facilitate folding of select group of cellular proteins that are crucial for cyto-protection and development of the parasites”.

Reviewer: The authors are encouraged to deposit the SAXS data in the SASBDB and link the accession number to the manuscript.

Authors Response: Thank you for this suggestion. We are considering depositing the data in the SASDB

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PLoS One. doi: 10.1371/journal.pone.0226657.r003

Decision Letter 1

Didier Picard

6 Apr 2020

Biophysical analysis of Plasmodium falciparum Hsp70-Hsp90 organizing protein (PfHop) reveals a monomer that is characterised by folded segments connected by flexible linkers

PONE-D-19-33340R1

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PLoS One. doi: 10.1371/journal.pone.0226657.r004

Acceptance letter

Didier Picard

9 Apr 2020

PONE-D-19-33340R1

Biophysical analysis of Plasmodium falciparum Hsp70-Hsp90 organising protein (PfHop) reveals a monomer that is characterised by folded segments connected by flexible linkers

Dear Dr. Shonhai:

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Supplementary Materials

S1 Fig. Purification of PfHop by ion exchange and size exclusion chromatography.

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S2 Fig. Mass spectrometry sequencing data for PfHop.

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S1 Table. PfHop secondary structure content.

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S1 Raw Images

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Biophysical analysis of Plasmodium falciparum Hsp70-Hsp90 organising protein (PfHop) reveals a monomer that is characterised by folded segments connected by flexible linkers

Stanley Makumire

Tawanda Zininga

Juha Vahokoski

Inari Kursula

Addmore Shonhai

Roles

Abstract

1. Introduction

2. Methods and materials

2.1 Materials

2.2 Expression and purification of recombinant PfHop

2.3 Investigation of the secondary structure of PfHop

2.4 Small-angle X-ray scattering analysis for PfHop shape determination

3. Results

3.1 Oligomeric state of recombinant PfHop

Fig 1. Determination of the oligomeric status of PfHop.

3.2 Analysis of the secondary structure of PfHop

Fig 2. Secondary structure analysis of PfHop.

Table 1. SAXS parameters for PfHop.

3.3 Low-resolution structure of PfHop in solution

Fig 3. SAXS analysis of PfHop.

4. Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Didier Picard

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Author response to Decision Letter 0

Decision Letter 1

Didier Picard

Roles

Acceptance letter

Didier Picard

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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