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

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 FP2(nose) and FP2(arm), 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 FP2(nose) 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 FP2(nose) and a conserved Glu outside this motif. The FP2(arm) motif is required for hemoglobinase activity. The structure reveals topographic features and a negative charge cluster surrounding FP2(arm) that suggest it may serve as an exo-site for hemoglobin binding. Motifs similar to FP2(nose) and FP2(arm) are found only in related plasmodial proteases, suggesting that they confer malaria-specific functions.

Fig. 1.

Structure of FP2–cystatin. (a) Overall structure of FP2–cystatin is depicted in stereo in a conversational orientation for papain-like cysteine proteases: with the L domain (Left), the R domain (Right), and the active site on the top facing forward. Cystatin is colored in orange, and FP2 protease is colored in green with the N-terminal extension and C-terminal insertion highlighted in pink and salmon, respectively. The L and R domains are highlighted by shades of blue and yellow, respectively. The shaded area also corresponds to the size of human cathepsin H (superimposed on FP2 in light blue). (b) Amino acid sequence of the mature proteases FP2, FP3, cathepsin K, cathepsin L, papain, and cruzain are aligned by using the ClustalW program (29). The secondary structure of FP2 is depicted above its sequence and compared with those of cathepsins K and L. Sequence motifs resembling FP2_nose and FP2_arm are highlighted in salmon and pink boxes. The helical folding motifs in FP2 and FP3 are highlighted in a pink shade. The two cysteines that form a disulfide bond not found in papain are highlighted with orange stars.

Fig. 2.

Functional FP2_nose in protein folding. (a) Interactions between FP2_nose and the rest of the protease are depicted in stereoview, with FP2_nose in pink and the protease core in green. All 17 amino acids in the nose region and residues from the protease core that make contact with the nose are shown in stick representation. Residues from the nose are marked in white labels with an N after their residue numbers. (b) Sequence alignment of plasmodial cysteine proteases from five different Plasmodium parasites (Plasmodium vivax, VX2 and VX3; Plasmodium knowlesi, KP2 and KP3; Plasmodium berghei, BP2; Plasmodium vinckei, VP2; and Plasmodium chabaudi, CP2) reveals a conserved 9-aa motif that may be important for protein folding as well as a KEA motif in the middle of the protease that interacts with the FP2_nose motif. (c) The catalytic activity of FP2_E120A is compared with wild-type FP2, using Z-Leu-Arg-AMC as a substrate as described (7). Results are presented as fluorescence unit (FU) per min. Error bars represent standard deviation from two assays, each performed in triplicate. (d) Data from CD analyses on FP2_E120A mutant and wild-type FP2. Absorbance between 200 and 270 nm was depicted for samples at various concentrations; 1 μM of E64 was added to both samples.

Fig. 3.

Structure and conservation of FP2 arm. (a) Putative arm regions from FP2-like cysteine proteases from five Plasmodium parasites are aligned by the ClustalW program. (b) 3F_o−2F_c electron density map of FP2_arm rendered at 1.2 σ is depicted in stereoview. Electron densities surrounding FP2_arm are contributed by FP2 residues from a neighboring symmetry mate (with carbons colored in white).

Fig. 4.

Active sites of FP2 versus papain complexed with protein and cathepsin V inhibitors. (a) FP2–cystatin, (b) papain–leupeptin (PDB ID code 1POP), and (c) cathepsin V-vinyl sulfone (PDB ID code 1FH0) are depicted in the same orientation. The catalytic Cys, His, and Asn are depicted in stick mode in green. Substrate-binding pockets are labeled P3 to P1′. The labels are positioned precisely in the same spacing to reflect the size differences of the substrate-binding pockets. Ile-68 is also depicted in stick mode to illustrate a slight protrusion in the S2 pocket of FP2.