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Structural insights into the inactive subunit of the apicoplast-localized caseinolytic protease complex of Plasmodium falciparum.

ABSTRACT: The ATP-dependent caseinolytic protease, ClpP, is highly conserved in bacteria and in the organelles of different organisms. In cyanobacteria, plant plastids, and the apicoplast of the genus Plasmodium, a noncatalytic paralog of ClpP, termed ClpR, has been identified. ClpRs are found to form heterocomplexes with ClpP resulting in a ClpRP tetradecameric cylinder having less than 14 catalytic triads. The exact role of ClpR in such a complex remains enigmatic. Here we describe the x-ray crystal structure of ClpR protein heptamer from Plasmodium falciparum (PfClpR). This is the first structure of a ClpR protein. The structure shows that the PfClpR monomer adopts a fold similar to that of ClpP, but has a unique motif, which we named the R-motif, forming a ? turn located near the inactive catalytic triad in a three-dimensional space. The PfClpR heptamer exhibits a more open and flat ring than a ClpP heptamer. PfClpR was localized in the P. falciparum apicoplast as is the case of PfClpP. However, biochemical and structural data suggest that, contrary to what has been observed in other organisms, PfClpP and PfClpR do not form a stable heterocomplex in the apicoplast of P. falciparum.

SUBMITTER: El Bakkouri M

PROVIDER: S-EPMC3542988 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Structural insights into the inactive subunit of the apicoplast-localized caseinolytic protease complex of Plasmodium falciparum.

El Bakkouri Majida M Rathore Sumit S Calmettes Charles C Wernimont Amy K AK Liu Kaiyin K Sinha Dipto D Asad Mohd M Jung Patrick P Hui Raymond R Mohmmed Asif A Houry Walid A WA

The Journal of biological chemistry 20121128 2

The ATP-dependent caseinolytic protease, ClpP, is highly conserved in bacteria and in the organelles of different organisms. In cyanobacteria, plant plastids, and the apicoplast of the genus Plasmodium, a noncatalytic paralog of ClpP, termed ClpR, has been identified. ClpRs are found to form heterocomplexes with ClpP resulting in a ClpRP tetradecameric cylinder having less than 14 catalytic triads. The exact role of ClpR in such a complex remains enigmatic. Here we describe the x-ray crystal str ...[more]

PMID: 23192353

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Project description: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 kcat/KM 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.

Dataset Information

Structural insights into the inactive subunit of the apicoplast-localized caseinolytic protease complex of Plasmodium falciparum.

Publications

Structural insights into the inactive subunit of the apicoplast-localized caseinolytic protease complex of Plasmodium falciparum.

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