Structural and Functional Characterization of Falcipain-2, a Hemoglobinase from the Malarial Parasite Plasmodium falciparum

Abstract

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.

BDA-410: A novel synthetic calpain inhibitor active against blood stage malaria

“…Recently, the crystal structure of FP2A complex with an irreversible inhibitor, iodoacetamide, was reported at 3.1A [35]. Interestingly, the crystal structure revealed the presence of four monomers of FP2A in the crystal, consistent with our recent demonstration that the recombinant FP2B, which is a paralog of FP2A, exists as a multimeric enzyme in solution [18,35].…”

“…Recently, the crystal structure of FP2A complex with an irreversible inhibitor, iodoacetamide, was reported at 3.1A [35]. Interestingly, the crystal structure revealed the presence of four monomers of FP2A in the crystal, consistent with our recent demonstration that the recombinant FP2B, which is a paralog of FP2A, exists as a multimeric enzyme in solution [18,35]. Clearly, additional structural studies on falcipains complexed with novel inhibitors will be needed to facilitate ongoing efforts to develop compounds that can selectively target falcipains and show relatively less inhibitory effects on host calpains and other cysteine proteases.…”

The Antimalarial Activities of Methylene Blue and the 1,4-Naphthoquinone 3-[4-(Trifluoromethyl)Benzyl]-Menadione Are Not Due to Inhibition of the Mitochondrial Electron Transport Chain

“…After reduction, these compounds can transfer single electrons to key oxidants of P. falciparum, e.g., to the hemoglobin catabolites methemoglobin and Fe(III)-protoporphyrin IX (22,24,26). The generated Fe(II) species are toxic to the parasite since Fe(II)-containing oxyhemoglobin is a poor substrate of the hemoglobinase falcipain-2 (27), and Fe(II)-protoporphyrin IX was shown to inhibit the crystallization of ␤-hematin in vitro (28). As a result of drug action, NA-DPH is consumed, and damaging Fe(II) species are constantly generated.…”

Centenary celebrations article

“…Further, the structure of falcipain indicates a protruding configuration for the motif, surrounded by a predominant negative charge , and may be charge residues are crucial for interaction with hemoglobin. It has also been demonstrated that falcipain-2 has relatively higher affinity for methemoglobin (kDa is 0.8 lM) than hemoglobin (kDa is 3.3 lM) (Hogg et al 2006). Based on this observation it has been suggested that several factors contribute to the formation of methemoglobin during malarial infection, including an acidic pH of plasmodial food vacuole, oxidative damage in RBC (Akompong et al 2000;Müller 2004), which causes an increase in methemoglobin content up to 20-42% in the plasmodial food vacuole.…”