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Structural basis for plasmepsin V inhibition that blocks export of malaria proteins to human erythrocytes

Nature Structural & Molecular Biology volume 22, pages 590–596 (2015)Cite this article

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Abstract

Plasmepsin V, an essential aspartyl protease of malaria parasites, has a key role in the export of effector proteins to parasite-infected erythrocytes. Consequently, it is an important drug target for the two most virulent malaria parasites of humans, Plasmodium falciparum and Plasmodium vivax. We developed a potent inhibitor of plasmepsin V, called WEHI-842, which directly mimics the Plasmodium export element (PEXEL). WEHI-842 inhibits recombinant plasmepsin V with a half-maximal inhibitory concentration of 0.2 nM, efficiently blocks protein export and inhibits parasite growth. We obtained the structure of P. vivax plasmepsin V in complex with WEHI-842 to 2.4-Å resolution, which provides an explanation for the strict requirements for substrate and inhibitor binding. The structure characterizes both a plant-like fold and a malaria-specific helix-turn-helix motif that are likely to be important in cleavage of effector substrates for export.

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Figure 1: Structures of WEHI-842 and WEHI-916 and effect on plasmepsin V function.
Figure 2: WEHI-842 inhibits cleavage and export of PEXEL proteins to the P. falciparum–infected erythrocyte.
Figure 3: Schematics showing the structure of P. vivax plasmepsin V in complex with WEHI-842.
Figure 4: Disulfide-bond architecture for aspartic acid proteases similar to plasmepsin V (PMV) and structural alignment of P. vivax plasmepsin V with P. falciparum plasmepsin II (PfPMII; PDB 1LF4).
Figure 5: Protein-ligand interactions across the active site of P. vivax plasmepsin V in complex with WEHI-842.

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Acknowledgements

We thank the Red Cross Blood Service (Melbourne, Australia) for supply of blood and the Australian Synchrotron and Commonwealth Scientific and Industrial Research Organisation Crystallization Facility. This work was supported by the Victorian State Government Operational Infrastructure Support and Australian Government National Health and Medical Research Council (NHMRC) Independent Research Institute Infrastructure Support Scheme, the Australian Cancer Research Foundation and the NHMRC (grants 1057960 (A.F.C.), 637406 (A.F.C.) and 1010326 (J.A.B.)). We thank the University of Melbourne for the provision of an Australian Postgraduate Award to M.G. A.F.C. is supported as a Howard Hughes International Scholar, P.E.C. is supported as an National Health and Medical Research Council of Australia Senior Research Fellow, and J.A.B. is supported as an Australian Research Council Queen Elizabeth II Fellow. We thank G. Lessene for useful discussions and Jacobus Pharmaceuticals for providing WR99210.

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Author notes

  1. Anthony N Hodder, Brad E Sleebs and Peter E Czabotar: These authors contributed equally to this work.

Authors and Affiliations

  1. Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Anthony N Hodder, Brad E Sleebs, Peter E Czabotar, Michelle Gazdik, Yibin Xu, Matthew T O'Neill, Sash Lopaticki, Thomas Nebl, Tony Triglia, Kym Lowes, Justin A Boddey & Alan F Cowman

  2. Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia

    Anthony N Hodder, Brad E Sleebs, Peter E Czabotar, Michelle Gazdik, Justin A Boddey & Alan F Cowman

  3. La Trobe Institute for Molecular Science, La Trobe University, Victoria, Australia

    Brian J Smith

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Contributions

A.N.H., B.E.S., J.A.B. and A.F.C. designed the experiments and wrote the manuscript. A.N.H. expressed, characterized, purified and crystallized plasmepsin V. B.E.S. and M.G. designed and synthesized WEHI-842. T.T. provided expert advice and supervision with respect to the molecular biology. P.E.C. collected X-ray data and solved and built the structure. P.E.C., A.N.H., B.J.S. and Y.X. did structural analysis of plasmepsin V with bound WEHI-842. B.J.S. and A.N.H. obtained enzyme kinetic data. M.T.O'N., S.L. and J.A.B. obtained biological data on the effect of WEHI-824. K.L. obtained surface plasmon resonance data. T.N. performed and analyzed proteomic experiments.

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Correspondence to Justin A Boddey or Alan F Cowman.

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Integrated supplementary information

Supplementary Figure 1 Expression and purification of functional P. vivax and P. falciparum plasmepsin V.

(a) First panel, cleavage of fusion tag from recombinant P. vivax plasmepsin V. Lane 1, P. vivax plasmepsin V prior to TEV (tobacco etch virus protease) cleavage, Lane 2, TEV, Lane 3, P. vivax plasmepsin V/TEV digest and Lane 4, control digest (no plasmepsin V). RD, reducing sample buffer. ND, non-reducing sample buffer. Cleaved tag is present at 20 kDa. Second panel, size exclusion chromatography (SEC) purification of monomeric P. vivax plasmepsin V. 1, 2 and 3 show key fractions from elution profile. Third panel, purified recombinant P. vivax plasmepsin V under reducing and non-reducing conditions. Fourth panel, purified inactive recombinant P. vivax plasmepsin V with mutation of D>N in the active site under reducing and non-reducing conditions. Fifth panel, Purified recombinant P. falciparum plasmepsin V under reducing and non-reducing conditions. (b) Partial sequence alignment of P. vivax and P. falciparum plasmepsin V. P. vivax plasmepsin V expressed from the prosequence (R35) to the C-terminus of the enzyme domain (R476). P. falciparum plasmepsin V was expressed with the five most C-terminal residues of the prosequence adjacent prior to the enzyme domain. (c) Activity of P. falciparum recombinant plasmepsin V using KAHRP PEXEL peptide with and without mutations of RL>A, R>K and L>I. (d) Activity of P. vivax plasmepsin V using KAHRP PEXEL peptide (RTLAQ) and PEXEL mutations RL>A, R>K and L>I. (e) P. vivax plasmepsin V with D>N mutation is inactive. (f-k) Michaelis–Menten kinetics of P. vivax and P. falciparum plasmepsin V. (f) and (g) Rate of PEXEL peptide cleavage by plasmepsin V. P. falciparum (f) and P. vivax plasmepsin V (g). (h) and (i) Burk-Lineweaver plot of the velocity of plasmepsin V as a function of substrate concentration for P. falciparum (h) and P. vivax (i) plasmepsin V. (j) and (k) Kinetics of substrate cleavage for P. falciparum (j) P. vivax plasmepsin V (k). (l) Summary of P. falciparum and P. vivax plasmepsin V kinetic values.

Supplementary Figure 2 Surface plasmon resonance analysis of WEHI-916 and WEHI-842 and effect of WEHI-842 on global protein synthesis in P. falciparum.

(a) and (b) Representative Surface plasmon resonance sensorgrams showing direct binding kinetics of WEHI-916 (a) and WEHI-842 (b) to P. vivax plasmepsin V. Binding to plasmepsin V at each concentration is illustrated by coloured curves with fit curves overlaid in black. (c) Trophozoites treated with WEHI-842 for 3 hr show no defect in protein synthesis (35S Autoradiograph). HSP70 levels were analysed as a loading and viability control (middle panel). The effect on PfEMP3-GFP processing was also examined by immunoblot and black arrow is uncleaved protein, red arrow is signal peptidase cleaved protein and blue arrow is PEXEL cleaved protein. A GFP only remnant in the food vacuole is also indicated. (d) Schematic of PfEMP3-GFP expressed in transgenic parasites showing the positions of processing.

Supplementary Figure 3 Structural features for P. vivax plasmepsin V.

(a) Putty diagram illustrating differences in B-factors within the structure of P. vivax plasmepsin V. Increased B-factors are shown as increased thickness and colour transition (blue to red). (b) Schematic showing surface electrostatic potential for P. vivax plasmepsin V. Top left image shows a face of the molecule enabling a full frontal view of the substrate binding cleft region. The top right hand side image is rotated 90˚ clockwise showing a face of the molecule with a side view of the substrate-binding cleft. There is a large area of negative electrostatic potential predicted to be adjoining the edge of the cleft and at the bottom. The bottom right hand side image represents the back face of the molecule with the substrate binding cleft facing into the page. The bottom left hand side image is of the alternative side face of P. vivax plasmepsin V and is predicted to have an extensive area of positive electrostatic surface potential.

Supplementary Figure 4 Sequence alignments of plasmepsin V from various Plasmodium species.

Alignments were conducted using clustal w and presented using the boxshade server (http://www.ch.embnet.org/software/BOX_form.html). Pv = Plasmodium vivax, Pcy = Plasmodium cynomolgi, Pi = Plasmodium inui, Pk = Plasmodium knowlesi, Pf = Plasmodium falciparum, Pchch = Plasmodium chabaudi chabaudi, Pvv = Plasmodium vinckei vinckei, Pyy = Plasmodium yoelii yoelii and Pb = Plasmodium berghei.

Supplementary Figure 5 Alignment of P. vivax plasmepsin V and P. falciparum plasmepsin II sequences and potential secondary-structural Nap insert and Flap elements.

(a) The alignment P. vivax plasmepsin V and P. falciparum plasmepsin II was made using the ESPript program (http://espript.ibcp.fr). The sequence corresponding to the region of the enzyme domains with crystal structures have been compared. Although P. vivax plasmepsin V and P. falciparum plasmepsin II have poor sequence homology most of the secondary structural elements are maintained. PDB file reference for P. falciparum plasmepsin II was 1LF4. (b) Alignment and structure of the Nap insert of plasmepsin V for P. vinckei (Pvvpmv), P. berghei (Pbpmv), P. yoelii (Pyypmv), P. falciparum (Pfpmv), P. vivax (Pvpmv), P. cynomolgi (Pcypmv), P. inui (Pipmv), P. knowlesi (Pkpmv) showing strong homology. Lower alignment shows Theileria ovis (ThoASP5), Babesia bovis (BboASP5), P. vivax (Pvpmv), Nepenthus distillatoria (NdNEP1), Nepenthus distillatoria (NdNEP2), Toxoplasma gondii (TgASP5) and low homology in the Nap insert. (c) Alignment and structure of the P. vivax flap region from plasmepsin V and other related proteases. P. vinckei (Pvvpmv), P. inui (Pipmv), P. cynomolgi (Pcypmv), P. knowlesi (Pkpmv), P. falciparum (Pfpmv), P. berghei (Pbpmv), P. chabaudi (Pchchpmv), P. yoelii (Pyypmv), P. vivax (Pvpmv), Theileria ovis (ThoASP5), Babesia bovis (BboASP5), Toxoplasma gondii (TgASP5), Nepenthus distillatoria (NdNEP1). (d) Model of flap regulation for plasmepsin V.

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Hodder, A., Sleebs, B., Czabotar, P. et al. Structural basis for plasmepsin V inhibition that blocks export of malaria proteins to human erythrocytes. Nat Struct Mol Biol 22, 590–596 (2015). https://doi.org/10.1038/nsmb.3061

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