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_x). E2-BeF_x 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_i) and stabilized by cardiotonic steroids, including a low-affinity Mg²⁺ 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 pre-occlusion transitions leading to activation of the dephosphorylation reaction. We show that the Mg²⁺ 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_x-inhibited enzyme is also able to bind ADP/ATP and Na⁺. These results relate the E2-BeF_x complex to a transient K⁺- and ADP-sensitive E*P intermediate of the functional cycle of the Na⁺,K⁺-ATPase, prior to E2P

Na ⁺ ,K ⁺ -ATP 酶通过包括各种磷酸酶中间体的功能循环产生跨质膜的 Na ⁺和 K ⁺电化学梯度。然而，这些中间体的结构和功能以及金属氟化物如何模仿它们需要进一步研究。在这里，我们描述了由抑制剂氟化铍（表示为 E2-BeF _x）稳定的猪肾 Na ⁺、K ^{+ -ATP 酶的 4.0 Å 分辨率晶体结构和功能特性。}E2- _BeFx预计将模拟 E2P 磷酸酶的特性，但具有未知的离子和配体结合特征。该结构类似于通过从无机磷酸盐 (P _i ) 磷酸化获得并由强心类固醇稳定的 E2P 形式，包括靠近离子结合位点 II的低亲和力 Mg ^{2+位点。我们对浸泡在 Rb +}（K ⁺同系物）中的晶体进行异常傅立叶分析，然后进行低分辨率刚体细化（6.9-7.5 Å），揭示了导致去磷酸化反应激活的预闭塞跃迁。我们表明，Mg ²⁺位置表明在与 Na 后向外开放的 E2P 状态结合时初始 K ^{+识别和接受的位点+}释放。此外，通过结合和活性研究，我们发现 BeF _x抑制酶也能够结合 ADP/ATP 和 Na ⁺。这些结果将 E2-BeF _{x复合物与 Na} ⁺、K ⁺ -ATP 酶功能循环的瞬态 K ⁺ - 和 ADP 敏感 E*P 中间体联系起来，在 E2P 之前