National Institutes of Health/National Cancer Institute (NIH/NCI)
RO1 CA69202
米国
引用
ジャーナル: iScience / 年: 2021 タイトル: Cryo-electron microscopy structures of VCP/p97 reveal a new mechanism of oligomerization regulation. 著者: Guimei Yu / Yunpeng Bai / Kunpeng Li / Ovini Amarasinghe / Wen Jiang / Zhong-Yin Zhang / 要旨: VCP/p97 is an evolutionarily conserved AAA+ ATPase important for cellular homeostasis. Previous studies suggest that VCP predominantly exists as a homohexamer. Here, we performed structural and ...VCP/p97 is an evolutionarily conserved AAA+ ATPase important for cellular homeostasis. Previous studies suggest that VCP predominantly exists as a homohexamer. Here, we performed structural and biochemical characterization of VCP dodecamer, an understudied state of VCP. The structure revealed an apo nucleotide status that has rarely been captured, a tail-to-tail assembly of two hexamers, and the up-elevated N-terminal domains akin to that seen in the ATP-bound hexamer. Further analyses elucidated a nucleotide status-dependent dodecamerization mechanism, where nucleotide dissociation from the D2 AAA domains induces and promotes VCP dodecamerization. In contrast, nucleotide-free D1 AAA domains are associated with the up-rotation of N-terminal domains, which may prime D1 for ATP binding. These results therefore reveal new nucleotide status-dictated intra- and interhexamer conformational changes and suggest that modulation of D2 domain nucleotide occupancy may serve as a mechanism in controlling VCP oligomeric states.
名称: VCP/p97 / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: all 詳細: VCP/p97 was overexpressed and purified from E.coli BL21(DE3). Purified protein was treated with apyrase to remove prebound ADP and generate apo VCP/p97.
凍結剤: ETHANE / チャンバー内湿度: 70 % / チャンバー内温度: 293 K / 装置: GATAN CRYOPLUNGE 3 詳細: 3.5ul sample was applied to a lacy carbon grid coated with graphene oxide. 7 seconds blot with filter paper was performed using Gatan Cp3..
詳細
VCP/p97 was overexpressed and purified with E.coli BL21(DE3). Purified proteins were treated with apyrase to remove prebound ADP and generate apo VCP/p97.
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電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
特殊光学系
エネルギーフィルター - 名称: GIF Quantum LS / エネルギーフィルター - スリット幅: 20 eV
撮影
フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: SUPER-RESOLUTION / 撮影したグリッド数: 1 / 実像数: 807 / 平均露光時間: 8.0 sec. / 平均電子線量: 40.0 e/Å2 詳細: A frame rate of 5 frames per second, a dose rate of 7.6 eps and a total exposure of 8 seconds were used.
電子線
加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN
電子光学系
C2レンズ絞り径: 100.0 µm / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.7 mm
クラス数: 2 / ソフトウェア - 名称: cryoSPARC 詳細: Two major classes including a dodecamer and a hexamer class. This submission is reporting reconstruction result for the hexamer class.