EMDB-3231: Structure and function based design of Plasmodium-selective prote...

Basic information

• Entry: Database: EMDB / ID: EMD-3231
• Title: Structure and function based design of Plasmodium-selective proteasome inhibitors
• Map data: unsharpened and unfiltered 3D reconstruction of the Plasmodium falciparum 20S proteasome core with a ligand

Sample

• Sample: Plasmodium falciparum 20S proteasome core bound to a specific inhibitor
• Protein or peptide: proteasome 20S core
• Ligand: Mor-WLW vinyl sulphone (HET: 7F1)

• Keywords: proteasome / 20S / Plasmodium / malaria / inhibitor / drug design / cryo-EM

Function / homology

Function:

Cross-presentation of soluble exogenous antigens (endosomes) / Orc1 removal from chromatin / CDK-mediated phosphorylation and removal of Cdc6 / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / KEAP1-NFE2L2 pathway / UCH proteinases / Ub-specific processing proteases / Neddylation / Antigen processing: Ubiquitination & Proteasome degradation / MAPK6/MAPK4 signaling / ABC-family proteins mediated transport / AUF1 (hnRNP D0) binds and destabilizes mRNA / Neutrophil degranulation / proteasome core complex / proteasomal ubiquitin-independent protein catabolic process / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / proteasomal protein catabolic process / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / endopeptidase activity / hydrolase activity / nucleus / cytosol / cytoplasm

Domain/homology:

Proteasome subunit beta 1 / Proteasome subunit alpha 1 / Proteasome subunit beta 4 / Proteasome subunit beta 2 / Proteasome beta 3 subunit / Proteasome subunit alpha6 / Proteasome subunit alpha5 / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / Nucleophile aminohydrolases, N-terminal

Component:

Proteasome subunit beta / Proteasome subunit alpha type-2, putative / Proteasome subunit alpha type-3, putative / Proteasome subunit beta / Proteasome subunit beta type-6, putative / Proteasome subunit beta / Proteasome subunit beta / Proteasome subunit alpha type-6, putative / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit alpha type / Proteasome subunit beta / Proteasome subunit alpha type-1, putative / Proteasome subunit beta

• Biological species: Plasmodium falciparum (malaria parasite P. falciparum) / synthetic construct (others)
• Method: single particle reconstruction / cryo EM / Resolution: 3.6 Å
• Authors: Li H / O' Donoghue AJ / van der Linden WA / Xie SC / Yoo E / Foe IT / Tilley L / Craik CS / da Fonseca PCA / Bogyo M
• Citation: Journal: Nature / Year: 2016; Title: Structure- and function-based design of Plasmodium-selective proteasome inhibitors.; Authors: Hao Li / Anthony J O'Donoghue / Wouter A van der Linden / Stanley C Xie / Euna Yoo / Ian T Foe / Leann Tilley / Charles S Craik / Paula C A da Fonseca / Matthew Bogyo / ; Abstract: The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the β2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials, we observe growth inhibition synergism with low doses of this β2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a chemically tractable target that could be exploited by next-generation anti-malarial agents.

History

Deposition	Nov 4, 2015	-
Header (metadata) release	Dec 16, 2015	-
Map release	Feb 17, 2016	-
Update	Mar 2, 2016	-
Current status	Mar 2, 2016	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_3231.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: unsharpened and unfiltered 3D reconstruction of the Plasmodium falciparum 20S proteasome core with a ligand
• Voxel size: X=Y=Z: 1.04 Å

Density

Contour Level	By AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum	-10.650586130000001 - 15.98163986
Average (Standard dev.)	0.0 (±1.0)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin -128 -128 -128 Dimensions 256 256 256 Spacing 256 256 256
Cell	A=B=C: 266.24 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.04	1.04	1.04
M x/y/z	256	256	256
origin x/y/z	0.000	0.000	0.000
length x/y/z	266.240	266.240	266.240
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	-128	-128	-128
NC/NR/NS	256	256	256
D min/max/mean	-10.651	15.982	-0.000

Supplemental data

Sample components

Entire : Plasmodium falciparum 20S proteasome core bound to a specific inh...

• Entire: Name: Plasmodium falciparum 20S proteasome core bound to a specific inhibitor

Components

• Sample: Plasmodium falciparum 20S proteasome core bound to a specific inhibitor
• Protein or peptide: proteasome 20S core
• Ligand: Mor-WLW vinyl sulphone (HET: 7F1)

Supramolecule #1000: Plasmodium falciparum 20S proteasome core bound to a specific inh...

• Supramolecule: Name: Plasmodium falciparum 20S proteasome core bound to a specific inhibitor; type: sample / ID: 1000 / Number unique components: 2
• Molecular weight: Theoretical: 760 KDa

Macromolecule #1: proteasome 20S core

• Macromolecule: Name: proteasome 20S core / type: protein_or_peptide / ID: 1 ; Details: The sample protein components are deposited in the PlasmoDB (the Plasmodium genome resource); Oligomeric state: dimer / Recombinant expression: No
• Source (natural): Organism: Plasmodium falciparum (malaria parasite P. falciparum)
• Molecular weight: Theoretical: 760 KDa

Macromolecule #2: Mor-WLW vinyl sulphone (HET: 7F1)

• Macromolecule: Name: Mor-WLW vinyl sulphone (HET: 7F1) / type: ligand / ID: 2 / Recombinant expression: No
• Source (natural): Organism: synthetic construct (others)

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.1 mg/mL
• Buffer: pH: 7.5 / Details: 50mM Tris HCl, 5mM MgCl2, 1mM dithiotreitol
• Grid: Details: 1.2/1.3 Quantifoil, 300 mesh Cu grids, coated with freshly floated thin layer of carbon
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK III / Method: blot 2.5 seconds before plunging

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Details: Each exposure was recorded as 17 individual frames captured at a rate of 0.056 second/frame, each with an electron dose of 2.8 electrons/square angstrom. Data-set recorded using EPU software.
• Date: Sep 3, 2014
• Image recording: Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 1816 / Average electron dose: 48 e/Ų / Details: each image was recorded as 17 frames
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 75000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Details: The particles were selected automatically using Relion, followed by manual inspection for the removal of false positives and addition of false negatives
• CTF correction: Details: full recorded image
• Final reconstruction: Applied symmetry - Point group: C₂ (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: OTHER / Software - Name: Tigris, Spider, Imagic / Number images used: 97720

Atomic model buiding 1

Initial model

PDB ID:

1iru

• Details: The initial models of the Plasmodium falciparum 20S proteasome core subunits were obtained using the PHYRE 2 structure prediction server; model building was done using real space refinement in Coot and Phenix
• Refinement: Space: REAL / Protocol: FLEXIBLE FIT

Output model

PDB-5fmg:
Structure and function based design of Plasmodium-selective proteasome inhibitors