Characterizing Protin Protein Interaction in Malarial Proteases

Abstract

Malaria is one of the deadliest diseases affecting humans with ~409000 deaths reported in 2019 alone. The current treatment regimen which includes artemisinins and their combination therapies is being threatened with the rapid emergence of resistance in South-East Asia and their gradual geographical expansion. This necessitates the need for new antimalarial drug targets that are less prone to resistance. Malarial proteases play crucial roles in almost all biological processes and are mostly substrate-specific, making them excellent targets for drug intervention strategies. Cysteine proteases, specifically falcipains, are principally responsible for the degradation of hemoglobin, the principal source of nutrients for blood-stage plasmodium parasites. Our study, through a combination of bioinformatic and mutagenesis analysis, has identified a single amino acid within both falcipains, falcipain-2 (FP2) and falcipain-3 (FP3), responsible for mediating interactions with hemoglobin. This approach is beneficial as the identified residue lies at an exosite protruding away from the active site, thus would likely be less prone to drug pressure. Further, we characterized the interactions between falcipains and their natural macromolecular inhibitor, falstatin which plays crucial roles during the invasion of RBC and hepatocytes. Falstatin was earlier shown to be unique as compared to its homologues and that only a single loop is sufficient for the inhibition of falcipains. Our current study suggests that falstatin interacts with FP2 in a multimeric form with ten units of falstatin interacting with ten units of FP2 in a 1:1 stoichiometry. A model of FP2-falstatin is proposed and key residues that could participate in this oligomerization were identified. The recombinant expression of a member belonging to another important class of proteases, plasmepsin IX (PMIX) has been achieved. With the recent developments surrounding PMIX (and PMX), it is advantageous to standardize recombinant protein expression, which could s