Structural mapping of the ClpBATPases of Plasmodium falciparum: Targeting protein folding and secretion for antimalarial drug design

Abstract

Caseinolytic chaperones and proteases (Clp) belong to the AAA1 protein superfamily and are part of the protein quality control machinery in cells. The eukaryotic parasite Plasmodium falciparum, the causative agent of malaria, has evolved an elaborate network of Clp proteins including two distinct ClpB ATPases. ClpB1 and ClpB2 are involved in different aspects of parasitic proteostasis. ClpB1 is present in the apicoplast, a parasite-specific and plastid-like organelle hosting various metabolic pathways necessary for parasite growth. ClpB2 localizes to the parasitophorous vacuole membrane where it drives protein export as core subunit of a parasite-derived protein secretion complex, the Plasmodium Translocon of Exported proteins (PTEX); this process is central to parasite virulence and survival in the human host. The functional associations of these two chaperones with parasite-specific metabolism and protein secretion make them prime drug targets. ClpB proteins function as unfoldases and disaggregases and share a common architecture consisting of four domains-a variable N-terminal domain that binds different protein substrates, followed by two highly conserved catalytic ATPase domains, and a C-terminal domain. Here, we report and compare the first crystal structures of the N terminal domains of ClpB1 and ClpB2 from Plasmodium and analyze their molecular surfaces. Solution scattering analysis of the N domain of ClpB2 shows that the average solution conformation is similar to the crystalline structure. These Abbreviations: Pfal, Plasmodium falciparum; AAA1, ATPase associated with diverse activities; Clp, caseinolytic protease; PTEX, Plasmodium translocon of exported proteins; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane; GFP, green fluorescent protein; SAXS, small angle X-ray scattering; RMSD, root-mean-square deviation; ADP, atomic displacement parameters; SEC, size exclusion chromatography; Ec, Escherichia coli; Tth, Thermus thermophilus; Bsu, Bacillus subtilis; Vch, Vibrio cholera.Additional Supporting Information may be found in the online version of this article.Andrew P. AhYoung and Antoine Koehl contributed equally to this work. Published by Wiley-Blackwell. V C 2015 The Protein Society structures represent the first step towards the characterization of these two malarial chaperones and the reconstitution of the entire PTEX to aid structure-based design of novel anti-malarial drugs.

Structure of a putative ClpS N‐end rule adaptor protein from the malaria pathogen Plasmodium falciparum

“…In Plasmodium , all Clp proteins, except ClpB2/HSP101, are localized in the apicoplast, a unique organelle that is evolutionarily related to the chloroplasts of plants, and a defining feature of the Apicomplexa group of parasites. ClpB2 (also named HSP101) is located in the vacuole between the plasma membrane and the parasitophorous vacuole membrane (PVM) that surrounds the parasite within the infected host where it functions as core subunit of the Plasmodium translocon of exported proteins, PTEX [Fig. (A)].…”

Crystal structure of Mdm12 and combinatorial reconstitution of Mdm12/Mmm1 ERMES complexes for structural studies

“…Experimental conditions were as previously described [30,31]. Scattering curves for the complex model were calculated using CRYSOL [32] and pair distance distributions - P(r) - derived by Fourier inversion using GNOM [33] to estimate Dmax , the longest distance occurring in the particle, and RG , its radius of gyration.…”

Crossing the Vacuolar Rubicon: Structural Insights into Effector Protein Trafficking in Apicomplexan Parasites

“…In chemical shift perturbation (CSP) NMR experiments, the largest CSPs were observed for NTD residues M1–A17 (H1), G80–L91 (loop between H3–H4, H4), V106–V108 (loop between H4–H5), and L111 (H5) in Thermus thermophilus ( Tt ) ClpB [ 75 ]. When mapped on the high-resolution crystal structures of Pf -HSP101/ClpB2 N -terminal domains, this corresponds to a hydrophobic batch capable of binding a range of unfolded peptidic sequences [ 60 ] ( Figure 7 A). Furthermore, the NTD of Tt -ClpB has regulatory roles that include blocking the translocation channel in the absence of substrate and destabilizing client proteins upon binding, thus priming them for subsequent unfolding and disaggregation [ 75 ].…”