ジャーナル: Proc Natl Acad Sci U S A / 年: 2015 タイトル: Structural characterization of the interaction of Ubp6 with the 26S proteasome. 著者: Antje Aufderheide / Florian Beck / Florian Stengel / Michaela Hartwig / Andreas Schweitzer / Günter Pfeifer / Alfred L Goldberg / Eri Sakata / Wolfgang Baumeister / Friedrich Förster / 要旨: 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.
ダウンロード / ファイル: emd_3034.map.gz / 形式: CCP4 / 大きさ: 81.8 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
Reconstruction of the 26S proteasome in presence of Ubp6 and ubiquitin aldehyde
ボクセルのサイズ
X=Y=Z: 1.99 Å
密度
表面レベル
登録者による: 1.17 / ムービー #1: 1.17
最小 - 最大
-11.709941860000001 - 12.581256870000001
平均 (標準偏差)
0.00614737 (±0.297102)
対称性
空間群: 1
詳細
EMDB XML:
マップ形状
Axis order
X
Y
Z
Origin
0
0
0
サイズ
280
280
280
Spacing
280
280
280
セル
A=B=C: 557.2 Å α=β=γ: 90.0 °
CCP4マップ ヘッダ情報:
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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添付データ
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試料の構成要素
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全体 : 26S Proteasome from Saccharomyces cerevisiae in the presence of S...
全体
名称: 26S Proteasome from Saccharomyces cerevisiae in the presence of Saccharomyces cerevisiae Ubp6 and ubiquitin aldehyde
要素
試料: 26S Proteasome from Saccharomyces cerevisiae in the presence of Saccharomyces cerevisiae Ubp6 and ubiquitin aldehyde
タンパク質・ペプチド: 26S Proteasome
タンパク質・ペプチド: Ubp6
タンパク質・ペプチド: ubiqutin aldehyde
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超分子 #1000: 26S Proteasome from Saccharomyces cerevisiae in the presence of S...
超分子
名称: 26S Proteasome from Saccharomyces cerevisiae in the presence of Saccharomyces cerevisiae Ubp6 and ubiquitin aldehyde タイプ: sample / ID: 1000 / Number unique components: 3
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補正
詳細: micrograph
最終 再構成
想定した対称性 - 点群: C1 (非対称) / アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 9.5 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: xmipp / 使用した粒子像数: 53000