Crucial residues in falcipains that mediate hemoglobin hydrolysis

Abstract

Falcipain-2 (FP2) and falcipain-3 (FP3) constitute the major hemoglobinases of Plasmodium falciparum. Previous biochemical and structural studies have explained the mechanism of inhibition of these enzymes by small molecules. However, a residue-level protein-protein interaction (PPI) with its natural macromolecular substrate, hemoglobin is not fully characterized. Earlier studies have identified a short motif in the C-terminal of FP2, an

exosite protruding away from the active site, essential for hemoglobin degradation. Our structural and muta- genesis studies suggest that hemoglobin interacts with FP2 via specific interactions mediated by Glu185 and

Val187 within the C-terminal motif, which are essential for hemoglobin binding. Since FP3 is also a major he- moglobinase and essential for parasite survival, we further demonstrate its interactions with hemoglobin. Our

results suggest that Asp194 of FP3 is required for hemoglobin hydrolysis and residue-swap experiments confirmed that this position is functionally conserved between the two hemoglobinases. Residues involved in protein–-

protein interactions constitute important targets for drug-mediated inhibition. Targeting protein–protein in- teractions at exosites may likely be less susceptible to emergence of drug resistance and thus is a new field to

explore in malaria.