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 a transport mechanism 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 employed single-molecule fluorescence resonance energy transfer (smFRET) to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca2+-ATPase (LMCA1), an orthologue of eukaryotic Ca2+-ATPases. Using the smFRET approach we identify key intermediates with no known crystal structures, and our findings delineate reversible and an essentially irreversible step in the transport process wherein Ca2+ efflux by LMCA1 is rate limited by phosphoenzyme formation and resolved by ADP and Ca2+ release leading to an open E2P state.

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