title: Three-dimensional structure of renal Na,K-ATPase from cryo-electron microscopy of two-dimensional crystals.
abstract: The structure of Na, K-ATPase was determined by electron crystallography at 9.5 A from multiple small 2-D crystals induced in purified membranes isolated from the outer medulla of pig kidney. The density map shows a protomer stabilized in the E(2) conformation which extends approximately 65 A x 75 A x 150 A in the asymmetric unit of the P2 type unit cell. The alpha, beta, and gamma subunits were demonstrated in the membrane crystals with Western blotting and related to distinct domains in the density map. The alpha subunit corresponds to most of the density in the transmembrane region as well as the large hydrophilic headpiece on the cytoplasmic side of the membrane. The headpiece is divided into three separated domains, which are similar in overall shape to the domains of the calcium pump of the sarcoplasmic reticulum. One of these domains gives rise to a characteristic elongated projection onto the membrane plane while the putative nucleotide binding and phosphorylation domains form comparatively compact densities in the rest of the cytoplasmic part of the structure. Density on the extracellular face corresponds to the protein part of the beta subunit and is located as an extension of the transmembrane region perpendicular to the membrane plane. The structure of the lipid bilayer spanning part suggests the positions for the transmembrane helix from the beta subunit as well as the small gamma subunit present in this Na,K-ATPase. Two groups of ten helices from the catalytic alpha subunit corresponds to the remaining density in the transmembrane region. The present results demonstrate distinct similarities between the structure of the alpha subunit of Na,K-ATPase as determined here by cryo-electron microscopy and the reported X-ray structure of Ca-ATPase. However, conformational changes between the E(1) and E(2) forms are suggested by different relative positions of cytoplasmatic domains.
authors.affiliation: Karolinska Institutet Department of Biosciences, Center for Structural Biochemistry, Novum, Huddinge, S-141 57, Sweden. Hans.Hebert@csb.ki.se
authors.forename: H
authors.initials: H
authors.lastname: Hebert
authors.forename: P
authors.initials: P
authors.lastname: Purhonen
authors.forename: H
authors.initials: H
authors.lastname: Vorum
authors.forename: K
authors.initials: K
authors.lastname: Thomsen
authors.forename: A B
authors.initials: AB
authors.lastname: Maunsbach
mesh_terms: D000818:Animals; D015153:Blotting, Western; D020285:Cryoelectron Microscopy; D003460:Crystallization; D007679:Kidney Medulla / Q000201:enzymology*; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D015203:Reproducibility of Results; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D013552:Swine
chemical_list: D021122:Protein Subunits; D000254:Sodium-Potassium-Exchanging ATPase