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 Mg$^{2+}$ 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$^{2+}$ 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.

Published by American Soc. for Biochemistry and Molecular Biology, Bethesda, MD.

JBC papers in press 298(9), 102317 (2022). doi:10.1016/j.jbc.2022.102317