Understanding the complex formation of falstatin; an endogenous macromolecular inhibitor of falcipains.

Rahul Pasupureddy, Sonia Verma, Bharti Goyal, Akansha Pant, Ruby Sharma, Shruti Bhatt, Kapil Vashisht, Shailja Singh, Ajay K Saxena, Rajnikant Dixit, Soumyananda Chakraborti, Kailash C Pandey

International journal of biological macromolecules 2024 Apr

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Proteolytic activity constitutes a fundamental process essential for the survival of the malaria parasite and is thus highly regulated. Falstatin, a protease inhibitor of Plasmodium falciparum, tightly regulates the activity of cysteine hemoglobinases, falcipain-2 and 3 (FP2, FP3), by inhibiting FP2 through a single surface exposed loop. However, the multimeric nature of falstatin and its interaction with FP2 remained unexplored. Here we report that the N-terminal falstatin region is highly disordered, and needs chaperone activity (heat-shock protein 70, HSP70) for its folding. Protein-protein interaction assays showed a significant interaction between falstatin and HSP70. Further, characterization of the falstatin multimer through a series of biophysical techniques identified the formation of a falstatin decamer, which was extremely thermostable. Computational analysis of the falstatin decamer showed the presence of five falstatin dimers, with each dimer aligned in a head-to-tail orientation. Further, the falstatin C-terminal region was revealed to be primarily involved in the oligomerization process. Stoichiometric analysis of the FP2-falstatin multimer showed the formation of a heterooligomeric complex in a 1:1 ratio, with the participation of ten subunits of each protein. Taken together, our results report a novel protease-inhibitor complex and strengthens our understanding of the regulatory mechanisms of major plasmodium hemoglobinases. Copyright © 2024. Published by Elsevier B.V.

Citation

Rahul Pasupureddy, Sonia Verma, Bharti Goyal, Akansha Pant, Ruby Sharma, Shruti Bhatt, Kapil Vashisht, Shailja Singh, Ajay K Saxena, Rajnikant Dixit, Soumyananda Chakraborti, Kailash C Pandey. Understanding the complex formation of falstatin; an endogenous macromolecular inhibitor of falcipains. International journal of biological macromolecules. 2024 Apr;265(Pt 1):130420