National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01 GM108043
米国
Danish Council for Independent Research
DFF-8021-00161
デンマーク
引用
ジャーナル: Proc Natl Acad Sci U S A / 年: 2021 タイトル: Structural basis for potassium transport in prokaryotes by KdpFABC. 著者: Marie E Sweet / Casper Larsen / Xihui Zhang / Michael Schlame / Bjørn P Pedersen / David L Stokes / 要旨: KdpFABC is an oligomeric K transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the superfamily of K ...KdpFABC is an oligomeric K transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the superfamily of K transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryogenic electron microscopy. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC, and KdpF remain static. We observe a series of spherical densities that we assign as K or water and which define a pathway for K transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K transfer between alternative sites in KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K to the cytoplasm.
超分子 #1: KdpFABC in E1 state with VO4 and K+ in sample buffer
超分子
名称: KdpFABC in E1 state with VO4 and K+ in sample buffer タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: all 詳細: KdpFABC complex solublized in decyl maltopyranoside (DM) detergent.
詳細: VO4 was added to the protein after size-exclusion chromatography and incubated 1 hr prior to freezing
グリッド
モデル: UltrAuFoil R1.2/1.3 / 材質: GOLD / メッシュ: 300 / 支持フィルム - 材質: GOLD / 支持フィルム - トポロジー: HOLEY ARRAY / 支持フィルム - Film thickness: 50.0 nm / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 雰囲気: AIR 詳細: PELCO easiGlow Glow Discharge Cleaning System used for glow discharge with default settings.
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277 K / 装置: FEI VITROBOT MARK IV 詳細: 3 uL of protein mixture applied to grid. Blot time 4 seconds, blot force 0, no wait before plunging..
詳細
DM-solublized KdpFABC complex purified through SEC the same day as grid freezing. Peak fraction diluted to 4.5 mg/mL and complexed with VO4 for 1 hr at room temperature prior to application to grid.