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

Marlene Uglebjerg Fruergaard, Ingrid Dach, Jacob Lauwring Andersen, Mette Ozol, Azadeh Shasavar, Esben Meldgaard Høgh Quistgaard, Hanne Poulsen, Natalya Fedosova*, Poul Nissen*

*Corresponding author af dette arbejde

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

Abstract

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.

OriginalsprogEngelsk
Artikelnummer102317
TidsskriftJournal of Biological Chemistry
Vol/bind298
Nummer9
Antal sider12
ISSN0021-9258
DOI
StatusUdgivet - 2022

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