title: A structural rearrangement of the Na+/K+-ATPase traps ouabain within the external ion permeation pathway.
abstract: With the use of the energy of ATP hydrolysis, the Na+/K+-ATPase is able to transport across the cell membrane Na+ and K+ against their electrochemical gradients. The enzyme is strongly inhibited by ouabain and its derivatives, some that are therapeutically used for patients with heart failure (cardiotonic steroids). Using lanthanide resonance energy transfer, we trace here the conformational changes occurring on the external side of functional Na+/K+-ATPases induced by the binding of ouabain. Changes in donor/acceptor pair distances are mainly observed within the α subunit of the enzyme. To derive a structural model matching the experimental lanthanide resonance energy transfer distances measured with bound ouabain, we carried out molecular dynamics simulations with energy restraints applied simultaneously using a novel methodology with multiple non-interacting fragments. The restrained simulation, initiated from the X-ray structure of the E2(2K+) state, became strikingly similar to the X-ray structure of the sodium-bound state. The final model shows that ouabain is trapped within the external ion permeation pathway of the pump.
authors: Department of Biochemistry and Molecular Biology, The University of Chicago Gordon Center for Integrative Science, Chicago, IL 60637, USA.
authors: Jorge E
authors: JE
authors: Sánchez-Rodríguez
authors: Department of Physics, University of Illinois at Chicago, Chicago, IL 60607, USA.
authors: Fatemeh
authors: F
authors: Khalili-Araghi
authors: Molecular Neurophysiology Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Pablo
authors: P
authors: Miranda
authors: Department of Biochemistry and Molecular Biology, The University of Chicago Gordon Center for Integrative Science, Chicago, IL 60637, USA. Electronic address: roux@uchicago.edu.
authors: Benoît
authors: B
authors: Roux
authors: Molecular Neurophysiology Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: Holmgren@ninds.nih.gov.
authors: Miguel
authors: M
authors: Holmgren
authors: Department of Biochemistry and Molecular Biology, The University of Chicago Gordon Center for Integrative Science, Chicago, IL 60637, USA. Electronic address: fbezanilla@uchicago.edu.
authors: Francisco
authors: F
authors: Bezanilla
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D002463:Cell Membrane Permeability; D018360:Crystallography, X-Ray; D049832:Decapodiformes; D031541:Fluorescence Resonance Energy Transfer; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D010042:Ouabain / Q000737:chemistry* / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D028581:Lanthanoid Series Elements; D021122:Protein Subunits; D010042:Ouabain; D000244:Adenosine Diphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium
keywords: cardiotonic steroids; energy transfer; ion transport; membrane protein; sodium/potassium pump
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