Malaria is caused by protozoan erythrocytic parasites of the Plasmodium genus, with Plasmodium falciparum being the most dangerous and widespread disease-causing species. Falcipain-2 (FP-2) of P. falciparum is a papain-family (C1A) cysteine protease that plays an important role in the parasite life cycle by degrading erythrocyte proteins, most notably hemoglobin. Inhibition of FP-2 and its paralogues prevents parasite maturation, suggesting these proteins may be valuable targets for the design of novel antimalarial drugs, but lack of structural knowledge has impeded progress toward the rational discovery of potent, selective, and efficacious inhibitors. As a first step toward this goal, we present here the crystal structure of mature FP-2 at 3.1 Å resolution, revealing novel structural features of the FP-2 subfamily proteases including a dynamic ␤-hairpin hemoglobin binding motif, a flexible N-terminal ␣-helical extension, and a unique active-site cleft. We also demonstrate by biochemical methods that mature FP-2 can proteolytically process its own precursor in trans at neutral to weakly alkaline pH, that the binding of hemoglobin to FP-2 is strictly pH-dependent, and that FP-2 preferentially binds methemoglobin over hemoglobin. Because the specificity and proteolytic activity of FP-2 toward its multiple targets appears to be pH-dependent, we suggest that environmental pH may play an important role in orchestrating FP-2 function over the different life stages of the parasite. Moreover, it appears that selectivity of FP-2 for methemoglobin may represent an evolutionary adaptation to oxidative stress conditions within the host cell.

Malaria is one of the major infectious diseases in the world. Approximately 300-500 million people are infected and 1-3 million deaths occur annually 1. The multidrug resistance of the parasite and limitations with existing drugs (e.g., toxicity, side effects, high cost) 2 emphasize the urgent need for new drugs, ideally directed against potential new targets such as plasmodial proteases. These proteases are multifunctional: During the merozoite stage of the parasite they facilitate erythrocyte escape and subsequent reinvasion. In the trophozoite stage these enzymes are critical for the degradation of hemoglobin 3. The falcipains are cysteine proteases belonging to the papain family (C1A). The best-studied members of this group are falcipains-1,-2 and-3. Falcipain-2 and-3 degrade hemoglobin at acidic pH. In addition, falcipain-2 degrades cytoskeletal proteins at neutral pH and thus facilitates the release of the mature merozoites. There is experimental evidence that cysteine protease inhibitors such as E-64, fluoromethyl ketones or aziridine derivatives inhibit falcipain-2 at low micromolar concentrations, and thereby block the development of the parasite 4,5. However, a falcipain-specific inhibitor with in-vivo activity remains to be developed. It is expected that the elucidation of the structure of falcipain-2 will open new avenues for the development of antimalarial drugs with lowered toxicity and drug resistance. Towards this goal, we have crystallized recombinant falcipain-2 and determined its structure to ∼ 3.1 Å resolution. There are two major structural differences between papain and falcipain-2. First, the presence of a ∼ 20-residue N-terminal extension which is necessary for the proper folding of the mature protein 6. Interestingly, this sequence element is functionally conserved within the plasmodial cysteine proteases 6. Second, falcipain-2 contains a rather unique structural motif responsible for the binding of its natural substrate, hemoglobin 7 : a beta-hairpin located between the active-site residues His174 and Asn204 where it protrudes out of the core of the enzyme. On the basis of our results, we will discuss the interaction between falcipain and its substrate, hemoglobin. Additional experimental data on substrate binding and specificity will be presented.