The Na,K-ATPase in complex with beryllium fluoride mimics an ATPase phosphorylated state.

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.