Poster Presentation The 47th Lorne Conference on Protein Structure and Function 2022

Structural and functional characterization of an exported plasmodial J domain protein, PFE0055c and its specific interactions with PfHsp70-x   (#357)

Tanima Dutta 1 2 , Harpreet Singh 3 , Jason E. Gestwick 4 , Gregory L. Blatch 1 2 5
  1. THE UNIVERSITY OF NOTRE DAME, WA, AUSTRALIA, FREMANTLE, WA, Australia
  2. The Institute for Immunology and Infectious Diseases, Murdoch University, Perth, Western Australia, Australia
  3. Department of Bioinformatics, Hans Raj Mahila Maha Vidyalaya, Jalandhar, Punjab, India
  4. Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, USA
  5. Faculty of Health Sciences, Higher Colleges of Technology, Sharjah, United Arab Emirates

Malaria accounts for high fatality rates of people and Plasmodium falciparum is known to be most virulent species of the malaria parasite. At the molecular level, survival of the parasite within the human host is proposed to require specialized proteins that provide protection during febrile episodes and the remodeling phase of host erythrocytes. This protection is mediated by P. falciparum heat shock proteins (PfHsps) that function as molecular chaperones. PfHsp70 proteins are widely characterized and several of them are known to be critical for parasite survival and virulence. Changes in the structure and protein profile of plasmodial parasites are major hurdles to the development of effective anti-malarial drugs, and PfHsps represent potential new anti-malarial drug targets. The ATP-dependent chaperone activity of Hsp70 depends on the co-chaperone J domain protein (JDP). There are over 49 PfHsp40s, some of which are essential for parasite survival, and many proposed to be required for protein folding and trafficking. Although a number of exported PfJDPs have been reported, very little is known about their structure, function and co-chaperone properties. The exported PfJDP protein, PFA0660w, has been shown to stimulate the ATPase activity of the exported chaperone, PfHsp70-x. Furthermore, PFA0660w has been shown to associate with another exported PfJDP, PFE0055c, and PfHsp70-x in J-dots localized to the infected erythrocyte cytosol. Therefore, the present study aims to conduct a structural and functional characterization of the full length PFE0055c. Recombinant PFE0055c was successfully expressed and purified using affinity chromatography under denaturing conditions and shown to stimulate the ATPase activity of PfHsp70-x to a higher extent in comparison to PFA0660w. Both PFE0055C and PFA0660w did not significantly stimulate the ATPase activity of human Hsp70. Small molecule inhibitors of Hsp70 (benzothiazole rhodacyanines, JG231 and JG98) and JDPs (chalcone, C86) were employed to determine their effect on basal and PFE0055c-stimulated ATPase activity of PfHsp70-x. This study showed that JG231 and JG98 inhibited both the basal and PFE0055c-stimulated ATPase activity of PfHsp70-x. C86 only inhibited the PFE0055c-stimulated ATPase activity of PfHsp70-x, consistent with PFE0055c binding to PfHsp70-x through its J domain. Biophysical studies employing surface plasmon resonance including specific inhibitors for JDPs are planned to validate the chaperone-co-chaperone complex formation of PFE0055c, PFA0660w and PfHsp70-x.This research has provided further insight into the molecular basis of the interaction between these exported plasmodial chaperones, which could inform future anti-malarial drug discovery studies.