title: The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop.
abstract: Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.
authors.affiliation: Department of Pharmacology and Toxicology, University of Lausanne, 1005 Lausanne, Switzerland.
authors.forename: Oihana
authors.initials: O
authors.lastname: Capendeguy
authors.affiliation: Swiss Institute of Bioinformatics, University of Lausanne, 1005 Lausanne, Switzerland.
authors.forename: Justyna
authors.initials: J
authors.lastname: Iwaszkiewicz
authors.affiliation: Swiss Institute of Bioinformatics, University of Lausanne, 1005 Lausanne, Switzerland; Ludwig Institute for Cancer Research/Lausanne Branch, University of Lausanne, 1005 Lausanne, Switzerland; Centre Pluridisciplinaire d'Oncologie, University of Lausanne, 1005 Lausanne, Switzerland.
authors.forename: Olivier
authors.initials: O
authors.lastname: Michielin
authors.affiliation: Department of Pharmacology and Toxicology, University of Lausanne, 1005 Lausanne, Switzerland. Electronic address: Jean-Daniel.Horisberger@unil.ch.
authors.forename: Jean-Daniel
authors.initials: JD
authors.lastname: Horisberger
mesh_terms: D020816:Amino Acid Motifs / Q000502:physiology; D019943:Amino Acid Substitution; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D018360:Crystallography, X-Ray; D005260:Female; D008958:Models, Molecular; D020125:Mutation, Missense; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D014982:Xenopus laevis
chemical_list: C499075:Atp1a1 protein, rat; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase