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 A?
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.