National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01 GM108043
米国
European Research Council (ERC)
637372
Danish Council for Independent Research
DFF-8021-00161
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.
詳細: The beryllium fluoride in the buffer was added to the protein mixture containing all other buffer components in the form of a pre-incubated mixture, at a 1:10 volume, to produce the final ...詳細: The beryllium fluoride in the buffer was added to the protein mixture containing all other buffer components in the form of a pre-incubated mixture, at a 1:10 volume, to produce the final concentrations 2.5 mM BeSO4 and 10 mM NaF.
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 magnesium fluoride solution for >30 min at room temperature prior to application to grid.