<i>Plasmodium falciparum</i> Hop (PfHop) Interacts with the Hsp70 Chaperone in a Nucleotide-Dependent Fashion and Exhibits Ligand Selectivity

<div><p>Heat shock proteins (Hsps) play an important role in the development and pathogenicity of malaria parasites. One of the most prominent functions of Hsps is to facilitate the folding of other proteins. Hsps are thought to play a crucial role when malaria parasites invade their host cells and during their subsequent development in hepatocytes and red blood cells. It is thought that Hsps maintain proteostasis under the unfavourable conditions that malaria parasites encounter in the host environment. Although heat shock protein 70 (Hsp70) is capable of independent folding of some proteins, its functional cooperation with heat shock protein 90 (Hsp90) facilitates folding of some proteins such as kinases and steroid hormone receptors into their fully functional forms. The cooperation of Hsp70 and Hsp90 occurs through an adaptor protein called Hsp70-Hsp90 organising protein (Hop). We previously characterised the Hop protein from <i>Plasmodium falciparum</i> (PfHop). We observed that the protein co-localised with the cytosol-localised chaperones, PfHsp70-1 and PfHsp90 at the blood stages of the malaria parasite. In the current study, we demonstrated that PfHop is a stress-inducible protein. We further explored the direct interaction between PfHop and PfHsp70-1 using far Western and surface plasmon resonance (SPR) analyses. The interaction of the two proteins was further validated by co-immunoprecipitation studies. We observed that PfHop and PfHsp70-1 associate in the absence and presence of either ATP or ADP. However, ADP appears to promote the association of the two proteins better than ATP. In addition, we investigated the specific interaction between PfHop TPR subdomains and PfHsp70-1/ PfHsp90, using a split-GFP approach. This method allowed us to observe that TPR1 and TPR2B subdomains of PfHop bind preferentially to the C-terminus of PfHsp70-1 compared to PfHsp90. Conversely, the TPR2A motif preferentially interacted with the C-terminus of PfHsp90. Finally, we observed that recombinant PfHop occasionally eluted as a protein species of twice its predicted size, suggesting that it may occur as a dimer. We conducted SPR analysis which suggested that PfHop is capable of self-association in presence or absence of ATP/ADP. Overall, our findings suggest that PfHop is a stress-inducible protein that directly associates with PfHsp70-1 and PfHsp90. In addition, the protein is capable of self-association. The findings suggest that PfHop serves as a module that brings these two prominent chaperones (PfHsp70-1 and PfHsp90) into a functional complex. Since PfHsp70-1 and PfHsp90 are essential for parasite growth, findings from this study are important towards the development of possible antimalarial inhibitors targeting the cooperation of these two chaperones.</p></div

PfHop is heat-inducible.

<p>The expression of PfHop and PfHsp70-1 was investigated on total cell lysate isolated from parasites cultured <i>in vitro</i> at 37°C, 41°C or 43°C. Samples were taken from cultures that were left to grow at the respective temperature for either 1 hour. The cell lysate was resolved by SDS-PAGE and Western blot analyses using antibodies recognizing PfHop, PfHsp70-1 and glycophorin (loading control), respectively.</p

SPR analysis for the self-association of PfHop recombinant protein.

<p>Analysis for self-association of PfHop was conducted using SPR analysis with PfHop as both ligand (1 μg/mL) and analyte. Variable amounts of analyte (2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.125 μM) were passed onto the ligand immobilized chip. The PfHsp70-1 (PfHsp70-1_NBD) was immobilised on the GLC chip as negative control. The sensograms represent data from at least three independent assays conducted in the presence of 5 mM ATP (panel A), 5mM ADP (panel B) and absence of nucleotides (panel C). The rate constants <i>ka</i>, <i>kd</i> and <i>K</i>_<i>D</i> values of PfHop self-association were determined (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135326#pone.0135326.s003" target="_blank">S2 Table</a>). The relative affinities of PfHop self-association were determined and presented as bar graphs (panel D). The relative affinities were normalized to PfHop self-association in the absence of nucleotides (NN). Error bars obtained from the t-tests are indicated. We observed no significant difference in the self-association of PfHop in the presence of ADP and presence of ATP (*), (<i>p<0</i>.<i>005</i>). However, affinity for the self-association of PfHop in the absence of nucleotides was significantly higher than obtained in the presence of nucleotide (**), (<i>p<0</i>.<i>005</i>).</p

PfHop co-immunoprecipitates with PfHsp70-1 in the presence of either ATP or ADP.

<p>α-PfHop antibodies were used in the co-immunoprecipitation (Co-IP) step and α -PfHsp70-1 and α -PfHop antibodies were used for Western analysis (panel A and B), respectively. Lane 1, whole parasite lysate prior to co-IP; lane 2, Co-IP conducted in presence of 5 mM ADP/ATP.</p

SPR analysis for the interaction of PfHop with PfHsp70-1.

<p>To further validate the interaction between PfHop and PfHsp70-1, SPR analysis was conducted with PfHop and PfHsp70-1 alternated as ligand and analyte, respectively. Full length PfHsp70-1 (1 μg/mL) was immobilised on the GLC sensor chip and variable concentrations of PfHop (2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.125 μM) were passed over the immobilised chaperone. The assay was repeated using the ATPase subdomain of PfHsp70-1 (PfHsp70-1_NBD) as a negative control. The sensograms represent data from at least three independent assays conducted in the presence of 5 mM ATP (panel A), 5mM ADP (panel B) and absence of nucleotides (panel C). The rate constants <i>k</i>_<i>a</i>, <i>k</i>_<i>d</i> and <i>K</i>_<i>D</i> values of PfHop towards PfHsp70-1, and PfHop self-association were determined (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135326#pone.0135326.s003" target="_blank">S2 Table</a>). The relative affinities of PfHop towards PfHsp70-1 and PfHop self-association were determined and presented as bar graphs (panel D). The relative affinities were normalized to PfHop interaction with PfHsp70-1in the absence of nucleotides (NN). Error bars obtained from the t-tests are indicated. We observed no significant difference in the association of PfHop with PfHsp70-1 in the presence of ADP and absence of nucleotide (*), (<i>p<0</i>.<i>005</i>). However, affinity for the interaction between PfHop and PfHsp70-1 in the presence of ATP was significantly lower than observed in the absence of nucleotide/presence of ADP (**), (<i>p<0</i>.<i>005</i>).</p

Expression and purification of recombinant PfHop and PfHsp70-1.

<p>SDS-PAGE (top panel) representing the purification of PfHop (~ 66 kDa) (panel A); PfHsp70-1 (~ 75 kDa) (panel B) and its ATPase domain (~ 45 kDa) (panel C). Note, the recombinant proteins were attached to an N-terminal histidine tag. The recombinant proteins were over-expressed in <i>E</i>. <i>coli</i> XL1 Blue cells and purified by nickel-affinity chromatography. Lane M, molecular markers in kDa; Lane C; XL1 Blue cells transformed with pQE30 vector; lane 0, pre-induction sample; lane 1, 1 hour post-IPTG induction sample and lane 5; sample taken 5 hours post-IPTG induction; lane S, supernatant of the whole lysate; lane W, wash; lane E, elution. α-PfHsp70-1 polyclonal antibody (1:2000) was used to confirm the presence of PfHsp70-1 and its ATPase subdomain by Western blotting (lower panels). The arrows indicate bands corresponding to the respective recombinant proteins: PfHop (Hop); PfHsp70-1 (70–1); and the ATPase subdomain of PfHsp70-1 (70-1A).</p