Journal: Proc Natl Acad Sci U S A / Year: 2016 Title: WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity. Authors: Qiuhong Li / Leifu Chang / Shintaro Aibara / Jing Yang / Ziguo Zhang / David Barford / Abstract: The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin-RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for ...The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin-RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for destruction via the ubiquitin proteasome system. The APC/C assembly comprises two scaffolding subcomplexes: the platform and the TPR lobe that together coordinate the juxtaposition of the catalytic and substrate-recognition modules. The platform comprises APC/C subunits Apc1, Apc4, Apc5, and Apc15. Although the role of Apc1 as an APC/C scaffolding subunit has been characterized, its specific functions in contributing toward APC/C catalytic activity are not fully understood. Here, we report the crystal structure of the N-terminal domain of human Apc1 (Apc1N) determined at 2.2-Å resolution and provide an atomic-resolution description of the architecture of its WD40 (WD40 repeat) domain (Apc1(WD40)). To understand how Apc1(WD40) contributes to APC/C activity, a mutant form of the APC/C with Apc1(WD40) deleted was generated and evaluated biochemically and structurally. We found that the deletion of Apc1(WD40) abolished the UbcH10-dependent ubiquitination of APC/C substrates without impairing the Ube2S-dependent ubiquitin chain elongation activity. A cryo-EM structure of an APC/C-Cdh1 complex with Apc1(WD40) deleted showed that the mutant APC/C is locked into an inactive conformation in which the UbcH10-binding site of the catalytic module is inaccessible. Additionally, an EM density for Apc15 is not visible. Our data show that Apc1(WD40) is required to mediate the coactivator-induced conformational change of the APC/C that is responsible for stimulating APC/C catalytic activity by promoting UbcH10 binding. In contrast, Ube2S activity toward APC/C substrates is not dependent on the initiation-competent conformation of the APC/C.
History
Deposition
Jul 7, 2016
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Header (metadata) release
Oct 5, 2016
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Map release
Oct 5, 2016
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Update
Aug 2, 2017
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Current status
Aug 2, 2017
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_4047.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel size
X=Y=Z: 1.36 Å
Density
Contour Level
By AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum
-0.18531242 - 0.3971233
Average (Standard dev.)
0.0022356252 (±0.016142555)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
-131
-131
-131
Dimensions
264
264
264
Spacing
264
264
264
Cell
A=B=C: 359.04 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
1.36
1.36
1.36
M x/y/z
264
264
264
origin x/y/z
0.000
0.000
0.000
length x/y/z
359.040
359.040
359.040
α/β/γ
90.000
90.000
90.000
MAP C/R/S
1
2
3
start NC/NR/NS
-131
-131
-131
NC/NR/NS
264
264
264
D min/max/mean
-0.185
0.397
0.002
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Supplemental data
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Sample components
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Entire : Ternary complex of APC/C-Cdh1-Hsl1 without Apc1 WD40 domain
Entire
Name: Ternary complex of APC/C-Cdh1-Hsl1 without Apc1 WD40 domain
Components
Complex: Ternary complex of APC/C-Cdh1-Hsl1 without Apc1 WD40 domain
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Supramolecule #1: Ternary complex of APC/C-Cdh1-Hsl1 without Apc1 WD40 domain
Supramolecule
Name: Ternary complex of APC/C-Cdh1-Hsl1 without Apc1 WD40 domain type: complex / ID: 1 / Parent: 0
Source (natural)
Organism: Homo sapiens (human)
Recombinant expression
Organism: Trichoplusia ni (cabbage looper) / Recombinant plasmid: pFU
Molecular weight
Theoretical: 1.2 MDa
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
cell
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Sample preparation
Concentration
0.12 mg/mL
Buffer
pH: 8 Component:
Concentration
Name
10.0 mM
HEPES
150.0 mM
NaCl
0.5 mM
TCEP
Details: Solutions were made fresh from powder and filtered to avoid microbial contamination.
Grid
Model: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 30.0 nm / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER Details: The grid was coated with continuous carbon film prior to use.
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III / Details: Blot for 5 seconds before plunging..
Details
Recombinant protein complex expressed and purified from insect cells. This sample was monodisperse.
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Electron microscopy
Microscope
FEI POLARA 300
Image recording
Film or detector model: FEI FALCON II (4k x 4k) / Detector mode: OTHER / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 4 / Number real images: 1729 / Average exposure time: 1.1 sec. / Average electron dose: 14.6 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Calibrated defocus max: 4.0 µm / Calibrated defocus min: 1.5 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
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Image processing
CTF correction
Software - Name: RELION (ver. 1.4)
Final reconstruction
Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 71724
Initial angle assignment
Type: OTHER / Software - Name: RELION (ver. 1.4)
Final angle assignment
Type: OTHER / Software - Name: RELION (ver. 1.4)
Final 3D classification
Number classes: 10 / Software - Name: RELION (ver. 1.4)
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Atomic model buiding 1
Refinement
Space: REAL / Protocol: FLEXIBLE FIT
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