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

• Published in: Nature (London) 2017-11, Vol.551 (7680), p.346-351
• Main Authors: Dyla, Mateusz, Terry, Daniel S., Kjaergaard, Magnus, Sørensen, Thomas L.-M., Lauwring Andersen, Jacob, Andersen, Jens P., Rohde Knudsen, Charlotte, Altman, Roger B., Nissen, Poul, Blanchard, Scott C.
• Format: Article
• Language: English
• Subjects: 631/45/173; 631/45/535; 631/57/2265; 96/33; Adenosine diphosphate; Adenosine Diphosphate - metabolism; Adenosine triphosphatase; Adenosine Triphosphate - metabolism; ATPases; Binding Sites; Biological transport; Ca2+-transporting ATPase; Calcium (extracellular); Calcium - metabolism; Calcium efflux; Calcium-Transporting ATPases - chemistry; Calcium-Transporting ATPases - metabolism; Cations; Cell membranes; Cells; Crystal structure; Efflux; Energy transfer; Enzyme kinetics; Fluorescence; Fluorescence Resonance Energy Transfer; Helices; Humanities and Social Sciences; Intermediates; Kinetics; Listeria; Listeria monocytogenes; Listeria monocytogenes - enzymology; Models, Molecular; Molecular biology; multidisciplinary; Phosphorylation; Physiological aspects; Physiological research; Potassium; Protein Conformation; Proteins; Science; Single Molecule Imaging; Transport processes
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature24296

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+ . Single-molecule fluorescence resonance energy transfer is used to identify the rate-limiting step and new intermediates in the conformational cycle of the Listeria monocytogenes calcium transporter LMCA1. FRET over enzyme dynamics P-type ATPases maintain electrochemical potentials across cell membranes by transporting cations against the concentration gradient. For example, ATP hydrolysis powers sarco/endoplasmic reticulum Ca( II )-ATPases (SERCAs), which drive Ca ion transport after Ca( II ) signalling events such as muscle contraction. Here, the authors use single-molecule fluorescence resonance energy transfer (FRET) methods to visualize the dynamics of a P-type Ca( II )-ATPase, LMCA1. Using this FRET-based approach to look at reaction cycle intermediates, they provide insights into the transport cycle and conformational dynamics of P-type ATPases at a single-molecule level that are not obtainable by structural or ensemble methods.