Decoding substrate recognition in malapain-2 through structural and mutational insights

Cysteine proteases of the falcipain (FP) family are essential for the survival and pathogenicity of <i>Plasmodium</i> parasites and represent promising targets for antimalarial drug development. These enzymes mediate haemoglobin degradation during the intraerythrocytic stage, providing nutrients and facilitating parasite growth. While FP-family proteases from <i>P. falciparum</i> (e.g., FP-2A and FP-3) are well-characterized, their orthologs in less-studied species like <i>P. malariae</i> remain poorly understood. Given the rising concern over drug-resistant malaria and mixed-species infections, targeting diverse FP-family ortholog enzymes in other human malaria parasites is a timely and promising therapeutic strategy. In this study, we investigated malapain-2 (MP-2), a cysteine protease from <i>P. malariae</i> belonging to the FP-2A/FP-3 subfamily, with the aim of characterizing its substrate specificity and structural features in comparison to FP-2A. Using biochemical assays, we found that MP-2 exhibited a distinct substrate preference, favouring arginine at the P2 position, unlike FP-2A which prefers hydrophobic residues. Site-directed mutagenesis and structural modelling revealed that differences in the S2 substrate-binding sub-pocket (key protease site) account for this specificity shift. Key mutations in MP-2 that mimic FP-2A residues altered substrate preference, confirming the role of specific residues in substrate accommodation. Docking and molecular dynamics simulations further supported these findings by revealing altered interaction networks within the binding pocket. Our results highlight the unique enzymatic properties of MP-2 and its potential as a species-specific drug target. This work expands our understanding of FP-family proteases across <i>Plasmodium</i> species and provides a foundation for the rational design of broad-spectrum or species-selective cysteine protease inhibitors.