Dynamics of P-type ATPase transport cycle revealed by single-molecule FRET

Mateusz Dyla, Daniel Terry, Magnus Kjærgaard, Thomas Lykke-Møller Sørensen, Jacob Lauwring Andersen, Jens Peter Andersen, Charlotte Rohde Knudsen, Roger Altman, Poul Nissen, Scott Blanchard

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67 Citations (Scopus)

Abstract

Phosphorylation-type (P-type) ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest that the transport mechanism is defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have used single-molecule fluorescence resonance energy transfer to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca 2+ -ATPase (LMCA1), an orthologue of eukaryotic Ca 2+ -ATPases. We identify key intermediates with no known crystal structures and show that Ca 2+ efflux by LMCA1 is rate-limited by phosphoenzyme formation. The transport process involves reversible steps and an irreversible step that follows release of ADP and extracellular release of Ca 2+.

Original languageEnglish
JournalNature
Volume551
Issue7680
Pages (from-to)346-351
Number of pages18
ISSN0028-0836
DOIs
Publication statusPublished - 16 Nov 2017

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