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The structure of the PA28–20S proteasome complex from Plasmodium falciparum and implications for proteostasis

Abstract

The activity of the proteasome 20S catalytic core is regulated by protein complexes that bind to one or both ends. The PA28 regulator stimulates 20S proteasome peptidase activity in vitro, but its role in vivo remains unclear. Here, we show that genetic deletion of the PA28 regulator from Plasmodium falciparum (Pf) renders malaria parasites more sensitive to the antimalarial drug dihydroartemisinin, indicating that PA28 may play a role in protection against proteotoxic stress. The crystal structure of PfPA28 reveals a bell-shaped molecule with an inner pore that has a strong segregation of charges. Small-angle X-ray scattering shows that disordered loops, which are not resolved in the crystal structure, extend from the PfPA28 heptamer and surround the pore. Using single particle cryo-electron microscopy, we solved the structure of Pf20S in complex with one and two regulatory PfPA28 caps at resolutions of 3.9 and 3.8 Å, respectively. PfPA28 binds Pf20S asymmetrically, strongly engaging subunits on only one side of the core. PfPA28 undergoes rigid body motions relative to Pf20S. Molecular dynamics simulations support conformational flexibility and a leaky interface. We propose lateral transfer of short peptides through the dynamic interface as a mechanism facilitating the release of proteasome degradation products.

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Fig. 1: Functional characterization of PfPA28.
Fig. 2: Crystal structure and sedimentation velocity analysis of PfPA28.
Fig. 3: SAXS analysis of PfPA28.
Fig. 4: The structure of the PfPA28−Pf20S complex.
Fig. 5: Proteasome gate control in Pf20S.
Fig. 6: Pivoting motion of the single-capped Pf20S−PfPA28 complex.

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Data availability

The coordinates and structure factors for PfPA28 were deposited in the PDB with accession code 6DFK. SAXS data and models were deposited in the Small Angle Scattering Biological Data Bank with accession code SASDES6. The coordinates for the uncapped, single PfPA28-capped and double PfPA28-capped Pf20S were deposited in the PDB with accession codes 6MUW, 6MUX and 6MUV, and the density maps were deposited in the Electron Microscopy Data Bank with accession codes EMD-9258, EMD-9259 and EMD-9257, respectively. The density map for the unstabilized PfPA28 single-capped complex was deposited in the Electron Microscopy Data Bank with accession code EMD-20073. The data that support the findings of this study are available from the corresponding authors on request.

Code availability

The computational codes or mathematical algorithms used in this study are available from the corresponding authors on request.

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Acknowledgements

We thank S. Tiash, The University of Melbourne, for technical support and advice and J. R. Beck, Iowa State University, for assistance with the generation of the pAIO-DHFR-PA28 vector. This work was supported by the National Health and Medical Research Council of Australia (grant nos. APP1092808, APP1072217) and the Global Health Innovation Technology Fund (grant no. GHIT T2015-134). M.D.W.G. is the recipient of an Australian Research Council Future Fellowship (project no. FT140100544). M.W.P. is a National Health and Medical Research Council of Australia Research Fellow (no. APP1117183). Funding from the Victorian Government Operational Infrastructure Support Scheme to St Vincent’s Institute is acknowledged. We thank the Medicines for Malaria Venture for ongoing support and the Australian Red Cross Blood Bank for the provision of human red blood cells and serum. We thank the Australian Synchrotron, part of the Australian Nuclear Science and Technology Organisation, for the provision of beamtime, and the beamline staff at the SAXS/WAXS and MX2 beamlines. This work made use of the ACRF Detector at the MX2 beamline. Initial crystallization screens were conducted at the CSIRO Collaborative Crystallisation Centre (www.csiro.au/C3). We thank the Bio21 Institute Advanced Microscopy Facility at The University of Melbourne.

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S.C.X., D.L.G. and T.Y. prepared protein samples and transfectants and undertook biochemical experiments. R.D.M., S.C.X. and M.D.W.G. performed the X-ray diffraction, SAXS and analytical ultracentrifugation experiments and refined the structures. E.H., A.P.L. and W.W. collected EM data and undertook reconstruction analyses. M.J.K. and C.J.M. performed the MD simulations. L.T., M.D.W.G., E.H., C.T. and L.R.D. conceived the study. L.T., M.D.W.G., N.J.S. and M.W.P. supervised experiments. All authors contributed to the writing of the manuscript.

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Correspondence to Michael D. W. Griffin or Leann Tilley.

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Supplementary Figs. 1–12, Tables 1–4, legends for Supplementary Videos and Supplementary References.

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Supplementary Video 1

Animation of a MD simulation of PfPA28.

Supplementary Video 2

Single-capped complex: eigenvectors 1 to 3.

Supplementary Video 3

Double-capped complex: eigenvectors 1 to 3.

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Xie, S.C., Metcalfe, R.D., Hanssen, E. et al. The structure of the PA28–20S proteasome complex from Plasmodium falciparum and implications for proteostasis. Nat Microbiol 4, 1990–2000 (2019). https://doi.org/10.1038/s41564-019-0524-4

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