The Plasmodium falciparum cysteine protease falcipain-2 captures its substrate, hemoglobin, via a unique motif

Abstract

Falcipain-2 (FP2) is a papain family cysteine protease and important hemoglobinase of erythrocytic Plasmodium falciparum parasites. Inhibitors of FP2 block hemoglobin hydrolysis and parasite development, suggesting that this enzyme is a promising target for antimalarial chemotherapy. FP2 and related plasmodial cysteine proteases have an unusual 14-aa motif near the C terminus of the catalytic domain. Recent solution of the structure of FP2 showed this motif to form a beta-hairpin that is distant from the enzyme active site and protrudes out from the protein. To evaluate the function of this motif, we compared the activity of the wild-type enzyme with that of a mutant lacking 10 aa of the motif (Delta10FP2). Delta10FP2 had nearly identical activity to that of the wild-type enzyme against peptide substrates and the protein substrates casein and gelatin. However, Delta10FP2 demonstrated negligible activity against hemoglobin or globin. FP2 that was inhibited with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (FP2E-64) formed a complex with hemoglobin, but Delta10FP2E-64 did not, indicating that the motif mediates binding to hemoglobin independent of the active site. A peptide encoding the motif blocked hemoglobin hydrolysis, but not the hydrolysis of casein. Kinetics for the inhibition of Delta10FP2 were very similar to those for FP2 with peptidyl and protein inhibitors, but Delta10FP2 was poorly inhibited by the inhibitory prodomain of FP2. Our results indicate that FP2 utilizes an unusual motif for two specific functions, interaction with hemoglobin, its natural substrate, and interaction with the prodomain, its natural inhibitor.

Fig. 1.

Structure of the C-terminal motif. (A) C-terminal motif sequences. Sequences of various proteases between the active site His and Asn (asterisks) were aligned with FP2 using the CLUSTALW program. The sequences deleted in the ^Δ10FP2 and ^Δ15FP2 mutants and that of the peptide used in this study are indicated. Amino acids forming the core of the motif in FP2 subfamily proteases are boxed. Amino acids identical to those of FP2 are red. (B) Structure of FP2. A surface diagram of FP2 shows positively (blue) and negatively (red) charged amino acids (S.X.W., K.C.P., J. Somoza, L. Brinen, P.S.S., P.J.R., and J.H.M., unpublished data). Key domains of the protease are labeled.

Fig. 2.

Hydrolysis of protein substrates by FP2 and ^Δ10FP2. (A) Gelatin. Equal concentrations (200 nM) of FP2 and ^Δ10FP2 were incubated with or without E-64, resolved by SDS/PAGE using nonreducing gels copolymerized with gelatin, incubated in reaction buffer, and then stained with Coomassie blue. (B) Casein. FP2 and ^Δ10FP2 at indicated concentrations were incubated with 3 μg of casein in the presence or absence of E-64 and reaction products were resolved by SDS/PAGE and staining with Coomassie blue. (C) FITC-casein. FITC-casein (10 μg) was incubated with different quantities of proteases at room temperature and fluorescence (FU) resulting from hydrolysis was measured. Error bars represent standard error of two independent measurements, each performed in duplicate.

Fig. 3.

Hydrolysis of hemoglobin. (A) Hydrolysis of hemoglobin and globin by FP2 and ^Δ10FP2. Proteins (3 μg) were incubated with 200 nM FP2 or ^Δ10FP2 in 20 mM Bis-Tris, pH 5.8, at room temperature, and reaction products were resolved by SDS/PAGE and staining with Coomassie blue. (B) Spectrophotometric assessment of hemoglobin hydrolysis. Native hemoglobin (30 μg) was incubated with FP2 or ^Δ10FP2 (200 nM) with the same buffer and conditions as in A and absorbance at 410 nm, indicative of disruption of hemoglobin, was monitored over time. The absorbance of each sample, expressed as percentage of the absorbance of hemoglobin with buffer only, was subtracted from 100 to yield percent hydrolysis. Error bars represent standard error of two independent measurements, each performed in duplicate.

Fig. 4.

Interaction between FP2 and hemoglobin. (A) Native gel analysis. Equal concentrations of FP2 or ^Δ10FP2 (400 nM) were incubated with E-64 to block their active sites and then incubated with 3 μg of hemoglobin (Hb). The proteins were resolved by 5% native PAGE and visualized with Coomassie blue. The locations of free enzyme, free hemoglobin, and the complex are indicated. (B) Plasmon resonance-based biomolecular interaction analysis. The hemoglobin-coupled flow cell was equilibrated, and indicated concentrations of FP2^E-64 and ^Δ10FP2^E-64 were injected over the sensor chip. The curves indicate surface plasmon resonance signals, in response units, representing binding to immobilized hemoglobin after subtraction of background, for the indicated concentrations of enzymes. (C) Stoichiometry analysis of FP2^E-64-hemoglobin by gel filtration chromatography. A curve based on molecular mass standards and the elution times for FP2^E-64, hemoglobin, and FP2^E-64-hemoglobin (arrows) are shown. The apparent molecular masses of FP2^E-64, hemoglobin, and FP2^E-64-hemoglobin were 12.5, 53.7, and 101 kDa, respectively, consistent with a 4:1 ratio of FP2 to hemoglobin in the complex.

Fig. 5.

Effect of the peptide encoding the motif on protein hydrolysis. (A) FITC-casein. The indicated amounts of peptide were preincubated with FITC-casein before addition of FP2. Hydrolysis of casein was measured as fluorescent units (FU). (B) Hemoglobin. Spectrophotometric assessment of hydrolysis of native hemoglobin by FP2 was assayed with and without the indicated amount of peptide for the indicated periods of time. For both graphs, error bars represent the standard error of two independent measurements, each performed in duplicate.