PEG400 regulates Falcipain 2 activity through an allosteric mechanism

The malarial parasite Plasmodium falciparum cleaves to host haemoglobin through a cascade of proteolytic enzymes. The cysteine protease, Falcipain-2 (FP2), plays an essential role in the process and is important for parasite survival, making it a potential drug target. However, similarities with host cysteine cathepsins hamper selective inhibition, thus necessitating detailed structural and functional characterisations of FP2. This study uncovers a new regulatory role for polyethylene glycol 400 (PEG400) on FP2 activity. PEG400 inhibits FP2 activity on small peptide substrates and azo-casein, while enhancing haemoglobin degradation, exerting a dual effect on FP2 catalysis. Mixed-type inhibition has been observed for PEG400 against small peptide substrates of FP2. This is consistent with the binding of PEG400 to the catalytic cleft, confirmed by fluorescence quenching and docking studies. Unlike typical nonspecific PEG-protein interactions, PEG400 adopts a fit within the catalytic region of FP2 and partially overlaps with leupeptin-binding sites, albeit with a lower affinity. Computational analysis further identifies a previously undescribed allosteric binding pocket of PEG400, which is further supported by in silico mutagenesis and molecular dynamics simulation studies. This pocket exhibits minimal conservation in human cathepsins, suggesting its selective potential. In contrast to this inhibitory role, biochemical assays revealed that PEG400 promotes haemoglobin proteolysis. Spectroscopic analyses further suggest that PEG400 alters haemoglobin structural dynamics to favour proteolysis. Interestingly, ENM-based normal mode analysis revealed that upon haemoglobin binding, PEG400 restricts the FP2 hinge-bending motion, improves FP2-haemoglobin proximity, and PEG400 is simultaneously dislodged from the active site, thereby promoting proteolysis. Altogether, these results reveal a previously undescribed mechanism of FP2 regulation, highlighting new therapeutic avenues.