ジャーナル: J Mol Biol / 年: 2021 タイトル: Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase. 著者: Milena Timcenko / Thibaud Dieudonné / Cédric Montigny / Thomas Boesen / Joseph A Lyons / Guillaume Lenoir / Poul Nissen / 要旨: P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of ...P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na/K-ATPase or Ca-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent.
平均露光時間: 1.5 sec. / 電子線照射量: 60 e/Å2 / フィルム・検出器のモデル: GATAN K3 (6k x 4k) / 撮影したグリッド数: 2 / 実像数: 6114
電子光学装置
エネルギーフィルター名称: GIF Bioquantum / エネルギーフィルタースリット幅: 20 eV
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解析
ソフトウェア
名称
バージョン
分類
phenix.real_space_refine
1.18rc7_3834
精密化
PHENIX
1.18rc7_3834
精密化
EMソフトウェア
ID
名称
バージョン
カテゴリ
詳細
2
EPU
画像取得
4
cryoSPARC
2
CTF補正
patchCTF
7
Coot
0.9-pre EL (ccpem)
モデルフィッティング
9
cryoSPARC
2
初期オイラー角割当
heterogeneousrefinement
10
cryoSPARC
2
最終オイラー角割当
non-uniform refinement
11
cryoSPARC
2
分類
heterogeneousrefinement
12
cryoSPARC
2
3次元再構成
non-uniform refinement
13
PHENIX
1.18rc7-3834
モデル精密化
CTF補正
タイプ: NONE
粒子像の選択
選択した粒子像数: 2423271
対称性
点対称性: C1 (非対称)
3次元再構成
解像度: 2.9 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 256001 / 対称性のタイプ: POINT
原子モデル構築
B value: 62 / 空間: REAL / Target criteria: correlation coefficient 詳細: Domains were rigid body fit into the density and manually adjusted and reconnected.