Catalytic Properties of Caseinolytic Protease Subunit of Plasmodium knowlesi and Its Inhibition by a Member of δ-Lactone, Hyptolide

Abstract

The caseinolytic protease (Clp) system plays an essential role in the protein homeostasis of the malaria parasite, particularly at the stage of apicoplast development. The inhibition of this protein is known to have a lethal effect on the parasite and is therefore considered an interesting avenue for antimalaria drugs discovery. The catalytic activity of the Clp system is modulated by its proteolytic subunit (ClpP), which belongs to the serine protease family member and is therefore extensively studied for further inhibitors development. Among many inhibitors, the group of β-lactone is known to be a specific inhibitor for ClpP. Nevertheless, other groups of lactones have never been studied. This study aims to characterize the catalytic properties of ClpP of Plasmodium knowlesi (Pk-ClpP) and the inhibition properties of a δ-lactone hyptolide against this protein. Accordingly, a codon-optimized synthetic gene encoding Pk-ClpP was expressed in Escherichia coli BL21(DE3) and purified under a single step of Ni²⁺-affinity chromatography, yielding a 2.20 mg from 1 L culture. Meanwhile, size-exclusion chromatography indicated that Pk-ClpP migrated primarily as homoheptameric with a size of 205 kDa. The specific activity of pure Pk-ClpP was 0.73 U µg^-1, with a catalytic efficiency k_cat/K_M of 0.05 µM^-1 s^-1, with optimum temperature and pH of 50 °C and 7.0-7.5, respectively. Interestingly, hyptolide, a member of δ-lactone, was shown to inhibit Pk-ClpP with an IC₅₀ value of 17.36 ± 1.44 nM. Structural homology modelling, secondary structure prediction, and far-UV CD spectra revealed that helical structures dominate this protein. In addition, the structural homology modeling showed that this protein forms a barrel-shaped homoheptamer. Docking simulation revealed that the inhibition was found to be a competitive inhibition in which hyptolide was able to dock into the catalytic site and block the substrate. The competitiveness of hyptolide is due to the higher binding affinity of this molecule than the substrate.

Keywords: Plasmodium knowlesi; antimalarial drug; caseinolytic protease; malaria; δ-lactone.

Figure 1

(a) Comparative amino acid sequence alignment of the ClpP from Plasmodium knowlesi (Pk-ClpP), P. falciparum (Pf-ClpP), Escherichia coli (Ec-ClpP), and human (Hs-ClpP). The catalytic triad of Ser-His-Asp is indicated by the arrow. (b) Secondary structure prediction by SOPMA. The motif of secondary structure is shown by the small letters below the sequence, where c, h, e, and t correspond to the random coil, α-helix, β-sheet (extended strand), and β-turn, respectively.

Figure 2

(a) The three-dimensional model of the ClpP from Plasmodium knowlesi (Pk-ClpP) in its oligomerization state. The monomeric form of Pk-ClpP is shown in green. (b) A close-up of the three-dimensional model of the monomeric form of ClpP from Plasmodium knowlesi. The N- and C-terminals of the protein were labeled as N- and C, respectively. The secondary structure arrangement was labeled with the number for clarity. The box is a close-up view of the triad catalytic site of Ser86-His112-Asp161. (c) Structural alignment between Pk-ClpP (green) and Pf-ClpP (red). The boxes in the dotted line indicate the structural differences between Pk-ClpP and Pf-ClpP. (d) Structural alignment between Pk-ClpP (green) and Ec-ClpP (purple). The boxes in the dotted line indicate the structural differences between Pk-ClpP and Ec-ClpP.

Figure 2

(a) The three-dimensional model of the ClpP from Plasmodium knowlesi (Pk-ClpP) in its oligomerization state. The monomeric form of Pk-ClpP is shown in green. (b) A close-up of the three-dimensional model of the monomeric form of ClpP from Plasmodium knowlesi. The N- and C-terminals of the protein were labeled as N- and C, respectively. The secondary structure arrangement was labeled with the number for clarity. The box is a close-up view of the triad catalytic site of Ser86-His112-Asp161. (c) Structural alignment between Pk-ClpP (green) and Pf-ClpP (red). The boxes in the dotted line indicate the structural differences between Pk-ClpP and Pf-ClpP. (d) Structural alignment between Pk-ClpP (green) and Ec-ClpP (purple). The boxes in the dotted line indicate the structural differences between Pk-ClpP and Ec-ClpP.

Figure 3

(a) Expression and solubility check of the ClpP from Plasmodium knowlesi (Pk-ClpP) by 15% SDS-PAGE. The M lane refers to the protein markers (kDa); Lanes 1 and 2 correspond to fractions before and after IPTG inductions, respectively; Lane 3 and 4 refer to pellet and soluble fractions obtained after cell lysis (sonication), respectively. (b) Purified Pk-ClpP under 15% SDS-PAGE. The P lane indicates the purified protein. The band corresponding to the Pk-ClpP protein is indicated by the arrow.

Figure 4

(a) Far-UV CD spectrum of ClpP from Plasmodium knowlesi (Pk-ClpP). (b) Unfolding curve of Pk-ClpP observed under the changes of CD value at 222 nm from 20–100 °C.

Figure 5

Elution profile of ClpP from Plasmodium knowlesi (Pk-ClpP) under size-exclusion chromatography (SEC). The elution profile of protein markers (β-amylase, alcohol dehydrogenase, albumin, carbonic anhydrase, and cytochrome C) are shown for comparison.

Figure 6

(a) Michaelis–Menten curve; (b) Lineweaver–Burk plot of the Pk-ClpP.

Figure 7

(a) Temperature- and (b) pH-dependent activities of Pk-ClpP. The highest activity was adjusted as 100%.

Figure 8

Relative activity of Pk-ClpP in the presence of various concentrations of hyptolide (Hyp) and a serine protease inhibitor PMSF (phenylmethylsulfonyl fluoride). The 100% relative activity was calculated from the activity without the inhibitors (not shown in the graph). The graph showed the relative activity in the presence of inhibitors with the concentration ranging from 1 nM (log 10⁰) to 500 nM (log10^2.69).

Figure 9

(a) Comparison of the binding position of hyptolide and substrate on the binding site of Pk-ClpP from the docking simulation. (b) The 2D map of the molecular interaction between Pk-ClpP and hyptolide. (c) The 2D map of the molecular interaction between Pk-ClpP and the substrate. Amino acids in blue and red round borders refer to basic and acid residues in contact. Exposure regions in the ligand are indicated by blue contour.

Figure 9

(a) Comparison of the binding position of hyptolide and substrate on the binding site of Pk-ClpP from the docking simulation. (b) The 2D map of the molecular interaction between Pk-ClpP and hyptolide. (c) The 2D map of the molecular interaction between Pk-ClpP and the substrate. Amino acids in blue and red round borders refer to basic and acid residues in contact. Exposure regions in the ligand are indicated by blue contour.