Structural basis for unique mechanisms of folding and hemoglobin binding by a malarial protease

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Abstract
Falcipain-2 (FP2), the major cysteine protease of the human malaria parasite Plasmodium falciparum, is a hemoglobinase and promising drug target. Here we report the crystal structure of FP2 in complex with a protease inhibitor, cystatin. The FP2 structure reveals two previously undescribed cysteine protease structural motifs, designated FP2nose and FP2arm, in addition to details of the active site that will help focus inhibitor design. Unlike most cysteine proteases, FP2 does not require a prodomain but only the short FP2nose motif to correctly fold and gain catalytic activity. Our structure and mutagenesis data suggest a molecular basis for this unique mechanism by highlighting the functional role of two Tyr within FP2nose and a conserved Glu outside this motif. The FP2arm motif is required for hemoglobinase activity. The structure reveals topographic features and a negative charge cluster surrounding FP2arm that suggest it may serve as an exo-site for hemoglobin binding. Motifs similar to FP2nose and FP2arm are found only in related plasmodial proteases, suggesting that they confer malaria-specific functions.