Mutation of GGMP Repeat Segments of Plasmodium falciparum Hsp70-1 Compromises Chaperone Function and Hop Co-Chaperone Binding

Abstract

Parasitic organisms especially those of the Apicomplexan phylum, harbour a cytosol localised canonical Hsp70 chaperone. One of the defining features of this protein is the presence of GGMP repeat residues sandwiched between α-helical lid and C-terminal EEVD motif. The role of the GGMP repeats of Hsp70s remains unknown. In the current study, we introduced GGMP mutations in the cytosol localised Hsp70-1 of Plasmodium falciparum (PfHsp70-1) and a chimeric protein (KPf), constituted by the ATPase domain of E. coli DnaK fused to the C-terminal substrate binding domain of PfHsp70-1. A complementation assay conducted using E. coli dnaK756 cells demonstrated that the GGMP motif was essential for chaperone function of the chimeric protein, KPf. Interestingly, insertion of GGMP motif of PfHsp70-1 into DnaK led to a lethal phenotype in E. coli dnaK756 cells exposed to elevated growth temperature. Using biochemical and biophysical assays, we established that the GGMP motif accounts for the elevated basal ATPase activity of PfHsp70-1. Furthermore, we demonstrated that this motif is important for interaction of the chaperone with peptide substrate and a co-chaperone, PfHop. Our findings suggest that the GGMP may account for both the specialised chaperone function and reportedly high catalytic efficiency of PfHsp70-1.

Keywords: GGMP repeats; Hop; Hsp70; Plasmodium falciparum; chaperone; malaria.

Figure 1

Multiple sequence alignment (MSA) of Hsp70s and design of GGMP mutants. (A) MSA of Plasmodium falciparum 3D7 Hsp70 (PfHsp70-1, NCBI accession number: XP_001349336.1); Plasmodium vivax (NCBI: XP_001614972.1); Plasmodium ovale curtisi (GenBank: SBS81157.1); Plasmodium malariae (NCBI: XP_028860418.1); Plasmodium knowlesi strain H (NCBI: XP_002258136.1); Plasmodium berghei ANKA Hsp70 (NCBI: XP_022712526.1); Cryptosporidium parvum Iowa II (NCBI: XP_625373.1); Cyclospora cayetanensis (NCBI: XP_022588287.1); Entamoeba histolytica (GenBank: AAA29102.1); Giardia lamblia ATCC 50,803 (GenBank: EDO80296.1); Leishmania donovani (UniprotKB: P17804); Toxoplasma gondii Hsp70 (UniprotKB: A0A125YXI9); Trypanosoma brucei HSP74 (UniprotKB: P11145); Homo sapiens heat shock cognate 71 kDa (Hsc70, NCBI: NP_006588.1); Homo sapiens Hsp70 protein 1A (HspA1A, NCBI: NP_005336.3); E. coli Hsp70 (DnaK, UniProtKB: A1A766.1); Mycobacterium tuberculosis Hsp70 (UniProtKB: P9WMJ9.1); Staphylococcus aureus HSP70 (GenBank: BAA06359.1); Saccharomyces cerevisiae HSP70 (Ssa1p, GenBank: AAC04952.1); Homo sapiens heat shock protein 70s (HspA14; NCBI: NP_057383.2), HspA6 (NCBI: NP_002146.2), HspA2 (NCBI: NP_068814.2), HspA4 (NCBI: NP_002145.3), HspA13 (NCBI: NP_008879.3), HspA12A (NCBI: NP_001317093.1), HspA12B (NP_001317093.2), HspA9 (NCBI, NP_004125.3). The black rectangle marks the GGMP repeat segment and the top panel highlights the overall residue conservation level. Residue numbering is based on PfHsp70-1. (B) Mutations were introduced at various positions within the GGMP repeat segment of PfHsp70-1 to generate the following derivatives: PfHsp70-1_G632, PfHsp70-1_G648, PfHsp70-_1G632-664, PfHsp70-1_∆G. The GGMP segment of PfHsp70-1 was inserted into DnaK to generate the derivative, “DnaK-_G”.

Figure 2

Superimposed three dimensional models of wild type SBDs of PfHsp70-1 and DnaK versus those of their GGMP variants. Superposition of the SBDβ and SBDα regions of PfHsp70-1 and DnaK against their respective GGMP variants was conducted: (A) PfHsp70-1 versus PfHsp70-1_G632, the insert represents zoomed segment highlights H-bonding variations; (B) PfHsp70-1 versus PfHsp70-1_G648, the zoomed SBD section with unique H-bonding is shown; (C) PfHsp70-1 and PfHsp70-1_G632-664, with the insert showing unique H-bonding variations in SBDβ; (D) PfHsp70-1 versus PfHsp70-1_ΔG structures and highlighted are structural variations within the SBD; and (E) DnaK superposed with DnaK-_G, insert highlights unique H-bonding pattern. The structures were visualized using Chimera.

Figure 3

Secondary structure analysis. (A) The CD spectra of native PfHsp70-1, PfHsp70-1_G632, PfHs70-1_G648 PfHsp70-1_G632-664, and PfHsp70-1_ΔG were presented as molar residue ellipticity (deg.cm².dmol⁻¹). (B) Temperature induced unfolding of the recombinant proteins was monitored by ellipticity measured at fixed wavelength of 222 nm as temperature was raised from 20 °C to 90 °C. Shown are: (C) CD spectra for DnaK and its mutant DnaK-_G; and (D) folded fraction of DnaK and DnaK-_G monitored at 222 nm. Dotted line represents the melting temperature for 50% of the Hsp70 or its respective variants. Relative folded fraction of each protein was determined at a given temperature relative to fully folded state of the protein observed at 20 °C.

Figure 4

GGMP mutations compromise basal ATPase and chaperone functions of PfHsp70-1. (A) ATPase activity of the GGMP variants relative to PfHsp70-1 (WT). The basal ATPase activities of PfHsp70-1, DnaK, and their GGMP variants were determined by monitoring the amount of Pi released determined by direct colorimetric readings conducted at 595 nm. (B) Heat induced aggregation suppression activities of PfHsp70-1 and its GGMP derivatives were monitored by exposing aggregation prone protein, MDH to heat stress at 51 °C in the presence of equimolar chaperone levels. The heat-induced aggregation of MDH was monitored spectroscopically at 360 nm. The assay was conducted in the absence of nucleotide (NN), or the presence of 5 mM ATP/ADP, respectively. (C) Complementation assay to determine the effect of the GGMP motifs on the function of chimeric protein, KPf, and DnaK. E. coli dnaK756 cells transformed with plasmid constructs expressing either KPf, its GGMP mutants; DnaK, and its GGMP insertion mutant, DnaK-_G, were incubated at the growth permissive temperature of 37 °C. Heat stress resilience of the cells was assessed by growing them 43.5 °C. Negative controls consisted of cells transformed with pQE60, pQE30 plasmid vectors and pQE60/KPf-V436F construct. Expression of the respective proteins was confirmed by Western blotting (“W”). Statistical analysis was carried out using one-way ANOVA at (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 5

GGMP mutation compromises peptide binding. The representative SPR sensograms for the interaction of PfHsp70-1 GGMP variants with the peptide ANNNMYRR. Assay was conducted in the absence of nucleotides (A), and the presence of either 5 mM ATP (B) or 5 mM ADP (C), respectively. The relative affinities of PfHsp70-1 and its GGMP variants for ANNNMYRR were determined as shown (D). The error bars indicate data generated from three assays conducted using independent Hsp70 protein preparations. Statistical significance was determined by two-way ANOVA (* p < 0.05, ** p < 0.01 and *** p < 0.001).

Figure 6

The GGMP motif of PfHsp70-1 modulates PfHop interaction. Representative SPR sensograms generated for assay monitoring interaction of PfHop and PfHsp70-1 GGMP variants are shown. Assay was conducted in the absence of nucleotides (A), and in the presence of either 5 mM ATP (B); or 5 mM ADP (C), respectively. The binding affinities of PfHop for PfHsp70-1 GGMP derivatives were normalized relative to the affinity of PfHop for wild type PfHsp70-1 as determined in the absence of nucleotides (D). The error bars represent data from three assays. Two-way ANOVA was used to determine the statistical significance at (** p < 0.01; *** p < 0.001).

Figure 7

Insertion of GGMP repeat residues into DnaK did not result in PfHop binding. Slot blot and ELISA were conducted to explore interaction of DnaK-_G with PfHop. The slot blot images and graphs generated from ELISA absorbance readings to explore interaction of PfHop with DnaK and its variant, DnaK-_G in the absence of nucleotides (A), and in the presence of either 5 mM ATP (B), or 5 mM ADP (C), respectively. The relative intensities for PfHop-DnaK-_G interaction were normalized relative to intensity of signal obtained for DnaK-PfHop at their highest amounts of the proteins for the assay conducted in the absence of nucleotides (D). The error bars represent data from three assays. Two-way ANOVA was used to determine the statistical significance at (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 8

Model highlighting the roles of the GGMP repeat residues of PfHsp70-1. PfHsp70-1 exhibits key functional features which are compromised upon removal or mutation of the GGMP repeat residues. The main structural defects leading to compromised function appear to include reorientation of the SBDβ loops and the C-terminal lid region. Reorientation of the SBDβ loops and the lid both adversely impact on substrate affinity and chaperone function.