Department of Molecular Biology and Genetics, DANDRITE - Nordic EMBL Partnership for Molecular Medicine, Aarhus University, Aarhus C, Denmark.
Department of Molecular Biology and Genetics, DANDRITE - Nordic EMBL Partnership for Molecular Medicine, Aarhus University, Aarhus C, Denmark; Department of Biomedicine, Aarhus University, Aarhus C, Denmark.
J Biol Chem. 2022 Sep;298(9):102317. doi: 10.1016/j.jbc.2022.102317. Epub 2022 Aug 2.
The Na,K-ATPase generates electrochemical gradients of Na and K across the plasma membrane via a functional cycle that includes various phosphoenzyme intermediates. However, the structure and function of these intermediates and how metal fluorides mimick them require further investigation. Here, we describe a 4.0 Å resolution crystal structure and functional properties of the pig kidney Na,K-ATPase stabilized by the inhibitor beryllium fluoride (denoted E2-BeF). E2-BeF is expected to mimic properties of the E2P phosphoenzyme, yet with unknown characteristics of ion and ligand binding. The structure resembles the E2P form obtained by phosphorylation from inorganic phosphate (P) and stabilized by cardiotonic steroids, including a low-affinity Mg site near ion binding site II. Our anomalous Fourier analysis of the crystals soaked in Rb (a K congener) followed by a low-resolution rigid-body refinement (6.9-7.5 Å) revealed preocclusion transitions leading to activation of the dephosphorylation reaction. We show that the Mg location indicates a site of initial K recognition and acceptance upon binding to the outward-open E2P state after Na release. Furthermore, using binding and activity studies, we find that the BeF-inhibited enzyme is also able to bind ADP/ATP and Na. These results relate the E2-BeF complex to a transient K- and ADP-sensitive E∗P intermediate of the functional cycle of the Na,K-ATPase, prior to E2P.
钠钾-ATP 酶通过一个功能循环在质膜两侧产生电化学梯度的钠和钾，该循环包括各种磷酸酶中间产物。然而，这些中间产物的结构和功能以及金属氟化物如何模拟它们需要进一步研究。在这里，我们描述了一个 4.0Å分辨率的晶体结构和猪肾钠钾-ATP 酶的功能特性，该酶由抑制剂铍氟化物（表示为 E2-BeF）稳定。E2-BeF 预计会模拟 E2P 磷酸酶的性质，但具有未知的离子和配体结合特性。该结构类似于通过无机磷酸盐（P）磷酸化并由强心甾稳定的 E2P 形式，包括离子结合位点 II 附近的一个低亲和力 Mg 结合位点。我们对浸泡在 Rb（一种 K 同系物）中的晶体进行的异常傅里叶分析，随后进行低分辨率刚体精修（6.9-7.5Å），揭示了导致去磷酸化反应激活的预阻塞转变。我们表明，Mg 位置表明，在 Na 释放后，结合到向外开放的 E2P 状态时，K 的初始识别和接受部位。此外，通过结合和活性研究，我们发现被 BeF 抑制的酶也能够结合 ADP/ATP 和 Na。这些结果将 E2-BeF 复合物与钠钾-ATP 酶功能循环中的一个短暂的 K 和 ADP 敏感的 E*P 中间产物联系起来，该中间产物在 E2P 之前。
