Cryoelectron microscopy of Na ⁺ ,K ⁺ -ATPase in the two E2P states with and without cardiotonic steroids

Abstract

Significance The E2P state of Na + ,K + -ATPase, in which the ATPase is phosphorylated and of low affinity to Na + with the extracellular gate opened, shows different biochemical properties depending on whether the phosphate is transferred from ATP in the forward reaction or from inorganic phosphate (P i ) in the backward reaction. We present here cryoelectron microscopy structures of Na + ,K + -ATPase in the two E2P states, explaining their different biochemical properties established a half century ago. The new electron microscopy maps show previously unseen structural features, including unexpected binding modes of cardiotonic steroids, specific and medically important inhibitors of the ATPase, and stabilization by ATP of the E2P state.

Cryo‐electron microscopy of Na+,K+‐ATPase reveals how the extracellular gate locks in the E2·2K+ state

“…In the E2P form, ATP itself bridges the A and N domains, thereby stabilising the cytoplasmic headpiece [20]. Here, in E2·2K + , we expect the opposite because ATP crosslinks the N domain to the P domain even in E2 [14].…”

“…relion 3.1 [19] was used for the following image processing. The cryo‐EM map of NKA in E2P with bound ouabain (PDB ID: 7WZ0, EMDB ID: 32900) [20] was used as the reference in the initial particle picking and 3D classification. The picked particles were subjected to 2D/3D classifications.…”

“…Cryo-electron microscopy of NKA from shark rectal gland in quasi-native lipid environment [20] was carried out for the E2Á2K + state, and 3D maps were obtained at 3.3 Å resolution (Fig. S1 and Table S1 for statistics).…”

Is the beta-subunit of Na,K-ATPase (NKA) a murzyme? Explaining the modulations by lithium ion, trans-dermal peptide, cardiotonic steroids, volatile anesthetics, arginine, dinitrophenol, and diverse antioxidants in NKA-aided neuro-musculo-electrophysiology

“…The recently reported structure of NKA from Sus scrofa (7WYT, Kanai et al, 2022) is analyzed herein (Figure 3). NKA is structurally and functionally analogous to H + -K + -ATPase and these enzymes belong to the P-type ATPase family.…”

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

“…Thus, a MgF x complex resembles an [K 2 ]E2∙P i state with occluded K + and noncovalently bound phosphate (P i ), whereas E1∙AlF 4 − ∙ADP contains three occluded Na + ions and represents an intermediate leading to the [Na 3 ]E1P-ADP phosphoenzyme ( 6 , 7 ). The BeF x complex of the Na + ,K + -ATPase ( 8 , 9 ) is structurally similar to the P i -induced (“backdoor” phosphorylated) E2P phosphoenzyme stabilized by cardiotonic steroids ( 10 , 11 ) and E2P ATP formed by ATP in the forward reaction ( 12 ).…”