Journal: Proc Natl Acad Sci U S A / Year: 2015 Title: Structural characterization of the interaction of Ubp6 with the 26S proteasome. Authors: Antje Aufderheide / Florian Beck / Florian Stengel / Michaela Hartwig / Andreas Schweitzer / Günter Pfeifer / Alfred L Goldberg / Eri Sakata / Wolfgang Baumeister / Friedrich Förster / Abstract: In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and ...In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediate-energy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation.
History
Deposition
Jun 1, 2015
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Header (metadata) release
Jul 8, 2015
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Map release
Jul 15, 2015
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Update
Aug 12, 2015
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Current status
Aug 12, 2015
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
#112 - Apr 2009 Oct and Sox Transcription Factors similarity (1)
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Map
File
Download / File: emd_3034.map.gz / Format: CCP4 / Size: 81.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Reconstruction of the 26S proteasome in presence of Ubp6 and ubiquitin aldehyde
Voxel size
X=Y=Z: 1.99 Å
Density
Contour Level
By AUTHOR: 1.17 / Movie #1: 1.17
Minimum - Maximum
-11.709941860000001 - 12.581256870000001
Average (Standard dev.)
0.00614737 (±0.297102)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
280
280
280
Spacing
280
280
280
Cell
A=B=C: 557.2 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
1.99
1.99
1.99
M x/y/z
280
280
280
origin x/y/z
0.000
0.000
0.000
length x/y/z
557.200
557.200
557.200
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-147
-147
-146
NX/NY/NZ
294
294
294
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
280
280
280
D min/max/mean
-11.710
12.581
0.006
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Supplemental data
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Sample components
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Entire : 26S Proteasome from Saccharomyces cerevisiae in the presence of S...
Entire
Name: 26S Proteasome from Saccharomyces cerevisiae in the presence of Saccharomyces cerevisiae Ubp6 and ubiquitin aldehyde
Components
Sample: 26S Proteasome from Saccharomyces cerevisiae in the presence of Saccharomyces cerevisiae Ubp6 and ubiquitin aldehyde
Protein or peptide: 26S Proteasome
Protein or peptide: Ubp6
Protein or peptide: ubiqutin aldehyde
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Supramolecule #1000: 26S Proteasome from Saccharomyces cerevisiae in the presence of S...
Supramolecule
Name: 26S Proteasome from Saccharomyces cerevisiae in the presence of Saccharomyces cerevisiae Ubp6 and ubiquitin aldehyde type: sample / ID: 1000 / Number unique components: 3
Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 5630 / Average electron dose: 45 e/Å2 / Details: Every image is the average of 7 aligned frames.
Tilt angle min
0
Tilt angle max
0
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
The particles were selected using an automatic selection program. Each physical 26S particles was considered as two particles for processing according to pseudo-C2 symmetry.
CTF correction
Details: micrograph
Final reconstruction
Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.5 Å / Resolution method: OTHER / Software - Name: xmipp / Number images used: 53000
Final two d classification
Number classes: 29100
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