Abstract
The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 are major hemoglobinases and potential antimalarial drug targets. Our previous studies demonstrated that these enzymes are equipped with specific domains for specific functions. Structural and functional analysis of falcipains showed that they have unique domains including a refolding domain and a hemoglobin binding domain. As with many proteases, falcipain-2 and falcipain-3 are synthesized as inactive zymogens. However, it is not known how these enzymes get activated for hemoglobin hydrolysis. In this study, we are presenting the first evidence that salt bridges and hydrophobic interactions are required for the auto activation of cysteine proteases of P.falciparum. To investigate the mechanism of activation of these enzymes, we expressed the wild type protein as well as different mutants in E.coli. Refolding was assessed by circular dichroism. Both CD and trans activation data showed that the wild type enzymes and mutants are rich in secondary structures with similar folds. Our study revealed that prodomain-mature domain of falcipain-2 and falcipain-3 interacts via salt bridges and hydrophobic interactions. We mutated specific residues of falcipain-2 and falcipain-3, and evaluated their ability to undergo auto processing. Mutagenesis result showed that two salt bridges (Arg 185 - Glu 221, Glu 210 - Lys 403) in falcipain-2, and one salt bridge (Arg 202-Glu 238) in falcipain-3, play crucial roles in the activation of these enzymes. Further study revealed that hydrophobic interactions present both in falcipain-2 (Phe214, Trp449 Trp 453) and falcipain-3 (Phe 231 Trp 457 Trp 461) also play important roles in the activation of these enzymes. Our results revealed the interactions involved in auto processing of two major hemoglobinases of malaria parasite.
Highlights
Malaria is a very important parasitic disease, and P.falciparum is the most virulent human malaria parasite causing 880,000 deaths per year worldwide [1]
We report here that salt bridges and hydrophobic interactions are crucial for auto activation of malarial cysteine proteases, falcipains
At 90 minutes one major band was observed resembling the mature domain of the enzyme, further confirmed by specific antibodies of FP2 (Fig. 2A)
Summary
Malaria is a very important parasitic disease, and P.falciparum is the most virulent human malaria parasite causing 880,000 deaths per year worldwide [1]. The development of other classes of effective anti-malarials, especially compounds that act against novel biochemical targets, is required. Short N-terminus extensions of the mature domains of falcipains mediate folding into active forms and a C-terminal insert in FP2 mediates its interaction with hemoglobin and subsequent hydrolysis [12,13,17]. It is not known how falcipains undergo processing upon reaching to the food vacuole. Falcipains are produced as inactive zymogens and likely undergo processing in the acidic food vacuole [8,9,21], where active enzymes hydrolyze hemoglobin. We report here that salt bridges and hydrophobic interactions are crucial for auto activation of malarial cysteine proteases, falcipains
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