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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
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.
References
Kirkman, L. A. et al. Antimalarial proteasome inhibitor reveals collateral sensitivity from intersubunit interactions and fitness cost of resistance. Proc. Natl Acad. Sci. USA 115, E6863–E6870 (2018).
Yoo, E. et al. Defining the determinants of specificity of Plasmodium proteasome inhibitors. J. Am. Chem. Soc. 140, 11424–11437 (2018).
Dogovski, C. et al. Targeting the cell stress response of Plasmodium falciparum to overcome artemisinin resistance. PLoS Biol. 13, e1002132 (2015).
Li, H. et al. Structure and function based design of Plasmodium-selective proteasome inhibitors. Nature 530, 233–236 (2016).
Bridgford, J. L. et al. Artemisinin kills malaria parasites by damaging proteins and inhibiting the proteasome. Nat. Commun. 9, 3801 (2018).
Harshbarger, W., Miller, C., Diedrich, C. & Sacchettini, J. Crystal structure of the human 20S proteasome in complex with carfilzomib. Structure 23, 418–424 (2015).
Huber, E. M. et al. Immuno- and constitutive proteasome crystal structures reveal differences in substrate and inhibitor specificity. Cell 148, 727–738 (2012).
Lin, G. et al. Inhibitors selective for mycobacterial versus human proteasomes. Nature 461, 621–626 (2009).
Löwe, J. et al. Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science 268, 533–539 (1995).
Groll, M. et al. Structure of 20S proteasome from yeast at 2.4 Å resolution. Nature 386, 463–471 (1997).
Choi, W. H. et al. Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation. Nat. Commun. 7, 10963 (2016).
Groll, M. et al. A gated channel into the proteasome core particle. Nat. Struct. Biol. 7, 1062–1067 (2000).
Voges, D., Zwickl, P. & Baumeister, W. The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem. 68, 1015–1068 (1999).
Mott, J. D. et al. PA28, an activator of the 20 S proteasome, is composed of two nonidentical but homologous subunits. J. Biol. Chem. 269, 31466–31471 (1994).
Respondek, D. et al. PA28 modulates antigen processing and viral replication during coxsackievirus B3 infection. PLoS ONE 12, e0173259 (2017).
Huang, L., Haratake, K., Miyahara, H. & Chiba, T. Proteasome activators, PA28γ and PA200, play indispensable roles in male fertility. Sci. Rep. 6, 23171 (2016).
Li, J., Powell, S. R. & Wang, X. Enhancement of proteasome function by PA28α; overexpression protects against oxidative stress. FASEB J. 25, 883–893 (2011).
Pickering, A. M. et al. The immunoproteasome, the 20S proteasome and the PA28αβ proteasome regulator are oxidative-stress-adaptive proteolytic complexes. Biochem. J. 432, 585–594 (2010).
Seifert, U. et al. Immunoproteasomes preserve protein homeostasis upon interferon-induced oxidative stress. Cell 142, 613–624 (2010).
Tanahashi, N. et al. Hybrid proteasomes. Induction by interferon-gamma and contribution to ATP-dependent proteolysis. J. Biol. Chem. 275, 14336–14345 (2000).
Cascio, P., Call, M., Petre, B. M., Walz, T. & Goldberg, A. L. Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes. EMBO J. 21, 2636–2645 (2002).
Sugiyama, M. et al. Spatial arrangement and functional role of α subunits of proteasome activator PA28 in hetero-oligomeric form. Biochem. Biophys. Res. Commun. 432, 141–145 (2013).
Li, H. et al. Validation of the proteasome as a therapeutic target in Plasmodium using an epoxyketone inhibitor with parasite-specific toxicity. Chem. Biol. 19, 1535–1545 (2012).
Boice, J. A. & Fairman, R. Structural characterization of the tumor suppressor p16, an ankyrin-like repeat protein. Protein Sci. 5, 1776–1784 (1996).
Ben-Saadon, R. et al. The tumor suppressor protein p16(INK4a) and the human papillomavirus oncoprotein-58 E7 are naturally occurring lysine-less proteins that are degraded by the ubiquitin system. Direct evidence for ubiquitination at the N-terminal residue. J. Biol. Chem. 279, 41414–41421 (2004).
Chen, X., Barton, L. F., Chi, Y., Clurman, B. E. & Roberts, J. M. Ubiquitin-independent degradation of cell-cycle inhibitors by the REGγ proteasome. Mol. Cell 26, 843–852 (2007).
Kobayashi, T., Wang, J., Al-Ahmadie, H. & Abate-Shen, C. ARF regulates the stability of p16 protein via REGγ-dependent proteasome degradation. Mol. Cancer Res. 11, 828–833 (2013).
Forster, A., Masters, E. I., Whitby, F. G., Robinson, H. & Hill, C. P. The 1.9 Å structure of a proteasome-11S activator complex and implications for proteasome-PAN/PA700 interactions. Mol. Cell 18, 589–599 (2005).
Zhang, Z. et al. Identification of an activation region in the proteasome activator REGα. Proc. Natl Acad. Sci. USA 95, 2807–2811 (1998).
Knowlton, J. R. et al. Structure of the proteasome activator REGα (PA28α). Nature 390, 639–643 (1997).
Petoukhov, M. V. et al. New developments in the ATSAS program package for small-angle scattering data analysis. J. Appl. Crystallogr. 45, 342–350 (2012).
Svergun, D. I., Petoukhov, M. V. & Koch, M. H. Determination of domain structure of proteins from X-ray solution scattering. Biophys. J. 80, 2946–2953 (2001).
Speed, M. A., Wang, D. I. & King, J. Specific aggregation of partially folded polypeptide chains: the molecular basis of inclusion body composition. Nat. Biotechnol. 14, 1283–1287 (1996).
Santner, A. A. et al. Sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression. Biochemistry 51, 7250–7262 (2012).
Zhang, Z., Realini, C., Clawson, A., Endicott, S. & Rechsteiner, M. Proteasome activation by REG molecules lacking homolog-specific inserts. J. Biol. Chem. 273, 9501–9509 (1998).
Blees, A. et al. Structure of the human MHC-I peptide-loading complex. Nature 551, 525–528 (2017).
Liu, X., Li, M., Xia, X., Li, X. & Chen, Z. Mechanism of chromatin remodelling revealed by the Snf2-nucleosome structure. Nature 544, 440–445 (2017).
Li, N. et al. Structure of the origin recognition complex bound to DNA replication origin. Nature 559, 217–222 (2018).
Martino, F. et al. RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex. Nat. Commun. 9, 1501 (2018).
De la Peña, A. H., Goodall, E. A., Gates, S. N., Lander, G. C. & Martin, A. Substrate-engaged 26S proteasome structures reveal mechanisms for ATP-hydrolysis-driven translocation. Science 362, eaav0725 (2018).
Forster, A., Whitby, F. G. & Hill, C. P. The pore of activated 20S proteasomes has an ordered 7-fold symmetric conformation. EMBO J. 22, 4356–4364 (2003).
Whitby, F. G. et al. Structural basis for the activation of 20S proteasomes by 11S regulators. Nature 408, 115–120 (2000).
Smith, D. M. et al. Docking of the proteasomal ATPases’ carboxyl termini in the 20S proteasome’s α ring opens the gate for substrate entry. Mol. Cell 27, 731–744 (2007).
Eisele, M. R. et al. Expanded coverage of the 26S proteasome conformational landscape reveals mechanisms of peptidase gating. Cell Rep. 24, 1301–1315 (2018).
Nakane, T., Kimanius, D., Lindahl, E. & Scheres, S. H. Characterisation of molecular motions in cryo-EM single-particle data by multi-body refinement in RELION. eLife 7, e36861 (2018).
Zhu, Y. et al. Structural mechanism for nucleotide-driven remodeling of the AAA-ATPase unfoldase in the activated human 26S proteasome. Nat. Commun. 9, 1360 (2018).
Tian, G. et al. An asymmetric interface between the regulatory and core particles of the proteasome. Nat. Struct. Mol. Biol. 18, 1259–1267 (2011).
da Fonseca, P. C., He, J. & Morris, E. P. Molecular model of the human 26S proteasome. Mol. Cell 46, 54–66 (2012).
Labun, K., Montague, T. G., Gagnon, J. A., Thyme, S. B. & Valen, E. CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res. 44, W272–W276 (2016).
Spillman, N. J., Beck, J. R., Ganesan, S. M., Niles, J. C. & Goldberg, D. E. The chaperonin TRiC forms an oligomeric complex in the malaria parasite cytosol. Cell Microbiol. 19, e12719 (2017).
Duraisingh, M. T., Triglia, T. & Cowman, A. F. Negative selection of Plasmodium falciparum reveals targeted gene deletion by double crossover recombination. Int. J. Parasitol. 32, 81–89 (2002).
Ganesan, S. M. et al. Yeast dihydroorotate dehydrogenase as a new selectable marker for Plasmodium falciparum transfection. Mol. Biochem. Parasitol. 177, 29–34 (2011).
Spillman, N. J., Dalmia, V. K. & Goldberg, D. E. Exported epoxide hydrolases modulate erythrocyte vasoactive lipids during Plasmodium falciparum infection. mBio 7, e01538-16 (2016).
Cabrita, L. D. et al. Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci. 16, 2360–2367 (2007).
Xie, S. C. et al. Target validation and identification of novel boronate inhibitors of the Plasmodium falciparum proteasome. J. Med. Chem. 61, 10053–10066 (2018).
Schuck, P. & Rossmanith, P. Determination of the sedimentation coefficient distribution by least-squares boundary modeling. Biopolymers 54, 328–341 (2000).
Ortega, A., Amorós, D. & García De La Torre, J. Prediction of hydrodynamic and other solution properties of rigid proteins from atomic- and residue-level models. Biophys. J. 101, 892–898 (2011).
Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).
Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D 67, 282–292 (2011).
Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D 69, 1204–1214 (2013).
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Cowtan, K. Recent developments in classical density modification. Acta Crystallogr. D 66, 470–478 (2010).
Cowtan, K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr. D 62, 1002–1011 (2006).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001).
Kirby, N. M. et al. A low-background-intensity focusing small-angle X-ray scattering undulator beamline. J. Appl. Crystallogr. 46, 1670–1680 (2013).
Kirby, N. et al. Improved radiation dose efficiency in solution SAXS using a sheath flow sample environment. Acta Crystallogr. D 72, 1254–1266 (2016).
Ryan, T. M. et al. An optimized SEC-SAXS system enabling high X-ray dose for rapid SAXS assessment with correlated UV measurements for biomolecular structure analysis. J. Appl. Crystallogr. 51, 97–111 (2018).
ScatterBrain v.2.82 (Australian Synchrotron SAXS/WAXS); https://archive.synchrotron.org.au/images/scatterBrainManual2.pdf
Franke, D. et al. ATSAS 2.8: a comprehensive data analysis suite for small-angle scattering from macromolecular solutions. J. Appl. Crystallogr. 50, 1212–1225 (2017).
Barberato, C., Koch, M. H. J., Molecular, E. & Outstation, H. CRYSOL - a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 28, 768–773 (1995).
ImageJ v.1.51m9 (NIH); https://imagej.nih.gov/ij/docs/examples/dot-blot/
Kimanius, D., Forsberg, B. O., Scheres, S. H. & Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016).
Scheres, S. H. Semi-automated selection of cryo-EM particles in RELION-1.3. J. Struct. Biol. 189, 114–122 (2015).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).
Terwilliger, T. C., Sobolev, O. V., Afonine, P. V. & Adams, P. D. Automated map sharpening by maximization of detail and connectivity. Acta Crystallogr. D 74, 545–559 (2018).
Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Headd, J. J. et al. Use of knowledge-based restraints in phenix.refine to improve macromolecular refinement at low resolution. Acta Crystallogr. D 68, 381–390 (2012).
Barad, B. A. et al. EMRinger: side chain–directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015).
Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).
Sali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).
Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).
Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).
Buchan, D. W., Minneci, F., Nugent, T. C., Bryson, K. & Jones, D. T. Scalable web services for the PSIPRED protein analysis workbench. Nucleic Acids Res. 41, W349–W357 (2013).
GraphPad Prism v.6.0; https://imagej.nih.gov/ij/docs/examples/dot-blot/
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.
Author information
Authors and Affiliations
Contributions
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.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Supplementary Information
Supplementary Figs. 1–12, Tables 1–4, legends for Supplementary Videos and Supplementary References.
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.
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41564-019-0524-4
This article is cited by
-
Structures revealing mechanisms of resistance and collateral sensitivity of Plasmodium falciparum to proteasome inhibitors
Nature Communications (2023)
-
PA28γ–20S proteasome is a proteolytic complex committed to degrade unfolded proteins
Cellular and Molecular Life Sciences (2022)
-
Cryo-EM of mammalian PA28αβ-iCP immunoproteasome reveals a distinct mechanism of proteasome activation by PA28αβ
Nature Communications (2021)
-
Expanding the role of proteasome homeostasis in Parkinson’s disease: beyond protein breakdown
Cell Death & Disease (2021)
-
Conformational maps of human 20S proteasomes reveal PA28- and immuno-dependent inter-ring crosstalks
Nature Communications (2020)