Journal Article

PUBDB-2022-07058

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png The Na$^+$,K$^+$-ATPase in complex with beryllium fluoride mimics an ATPase phosphorylated state

Fruergaard, M. U. ; Dach, I. ; Andersen, J. L. ; Ozol, M. ; Shahsavar, A. ; Quistgaard, E. M. ; Poulsen, H. ; Fedosova, N. U. (Corresponding author) ; Nissen, P. (Corresponding author)Extern*

2022
American Soc. for Biochemistry and Molecular Biology Bethesda, MD.

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Please use a persistent id in citations: doi:10.1016/j.jbc.2022.102317  doi:10.3204/PUBDB-2022-07058

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.

Note: ISSN 0021-9258 not unique: **2 hits**.

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Contributing Institute(s):

1. EMBL-User (EMBL-User)

Research Program(s):

1. 6G3 - PETRA III (DESY) (POF4-6G3) (POF4-6G3)

Experiment(s):

1. PETRA Beamline P14 (PETRA III)
2. PETRA Beamline P13 (PETRA III)

Appears in the scientific report 2022

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Database coverage:
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