National Health and Medical Research Council (Australia)
Australia
Australian Research Council
Australia
Citation
Journal: Nat Microbiol / Year: 2019 Title: The structure of the PA28-20S proteasome complex from Plasmodium falciparum and implications for proteostasis. Authors: Stanley C Xie / Riley D Metcalfe / Eric Hanssen / Tuo Yang / David L Gillett / Andrew P Leis / Craig J Morton / Michael J Kuiper / Michael W Parker / Natalie J Spillman / Wilson Wong / ...Authors: Stanley C Xie / Riley D Metcalfe / Eric Hanssen / Tuo Yang / David L Gillett / Andrew P Leis / Craig J Morton / Michael J Kuiper / Michael W Parker / Natalie J Spillman / Wilson Wong / Christopher Tsu / Lawrence R Dick / Michael D W Griffin / Leann Tilley / 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 ...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.
Name: 20S proteasome/PA28 complex. Details: Sample was a mixture of unbound 20S proteasome, 20S proteasome in complex with one PA28 cap, and 20S proteasome in complex with two PA28 caps. Number of components: 3
Name: 20S proteasome/PA28 complex. Details: Sample was a mixture of unbound 20S proteasome, 20S proteasome in complex with one PA28 cap, and 20S proteasome in complex with two PA28 caps. Recombinant expression: No
Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277.15 K / Humidity: 100 % / Details: wait time 0sec blot time 2sec blot force -1.
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Electron microscopy imaging
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
Imaging
Microscope: FEI TALOS ARCTICA
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 32 e/Å2 / Illumination mode: FLOOD BEAM
Lens
Magnification: 100000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - nm
Specimen Holder
Model: FEI TITAN KRIOS AUTOGRID HOLDER
Camera
Detector: GATAN K2 QUANTUM (4k x 4k)
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Image acquisition
Image acquisition
Number of digital images: 622 / Sampling size: 5 µm
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Image processing
Processing
Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 18893 / Details: Images were gain corrected
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