The three-dimensional structure of the Na,K-ATPase from electron microscopy.

Abstract

Abstract. The structure of Na,K-ATPase has been studied by electron microscopy and image reconstruction. A three-dimensional structure of this enzyme has been obtained to an overall resolution of 2.5 nm using data from specimens of negatively stained dimer sheets tilted through a range of angles +60 °.The reconstruction shows a complex mass distribution consisting of ribbons of paired molecules extending ,',,6.0 nm from the cytoplasmic side of the membrane. The molecular envelope consists of a massive "body" with "lobe" and "arm" structures projecting from it. The body has a columnar shape and is tilted with respect to the plane of the membrane. The region of interaction responsible for dimer formation is located between two bodies and is clearly visible in the reconstruction. It has been identified as a segment in the amino-terminal portion of the ct subunit. The arms that interconnect the ribbons are located close to the membrane and are most probably formed by the 13 subunits. NA,K-ATPaSe constitutes the ion pump that couples the hydrolysis of ATP to the transmembrane transport of sodium and potassium ions. It is primarily responsible for the establishment and maintenance of the gradients of Na + and K ÷ across the plasma membrane of eukaryotic cells. The transmembrane potential produced by this process is used by the cell to power other transport processes. The enzyme is built from two polypeptide chains (7,14,16,20), of which ¢t (Mr 110,000) is the catalytic subunit and 13 (mr 50,000-60,000) is a glycoprotein whose function has yet to be determined.The availability of purified specimens of Na,K-ATPase has allowed extensive studies of its various properties (for reviews see references 5, 10, 17, 32). The recent progress in structural studies of the enzyme has been particularly impressive. The amino acid sequence of ¢t (18, 34) and 13 (19,29,33) have been determined from various tissues. Furthermore, the discovery of crystallization schemes that produced regular arrays of the enzyme in membrane fragments (35) initiated structural studies by electron microscopy and image processing. Structural analyses of the enzyme in projection have been reported (11,23,42) as well as preliminary data on its three-dimensional structure (13,27,31). In this laboratory we have tentatively mapped the domains in the projection image of the enzyme that correspond to its two subunits (23, 25), and have found reliable crystallization methods to produce regular sheets of the enzyme in ap2 lattice (26).In this paper we present a three-dimensional structural study of Na,K-ATPase to an overall resolution of 2.5 nm. The reconstruction shows the structure of the enzyme on the cgtoplasmic side of the membrane, and reveals that it consists of tilted columns of mass interconnected by a complex set of bridges. These bridges can be identified with the bonds responsible for dimer formation and the subsequent ribbon-ribbon interaction. A segment of the amino-terminal of the ct subunit can be located within the structure and a tentative identific...

Molecular imaging of Na+,K+‐ATPase in purified kidney membranes

“…The Na÷,K+-ATPase molecules in kidney membranes (see section 2) have been well characterized by conventional biochemical techniques and high resolution EM [6][7][8][9][10][11]18]. Our specimens under EM showed membrane vesicles (Fig.…”

“…ld). Although the membrane-bound Na+,K ÷-ATPase molecules have been known to form two-dimensional crystals in the presence of vanadate or phosphate (Pi) and Mg 2÷ [6][7][8][9][10][11], the specimens used in the present study were not treated with these reagents but negatively stained with uranyl acetate. This procedure added mechanical strength to the samples, and made the images obtained from EM and AFM comparable.…”

“…In the case of membrane proteins, this task has typically proven to be a challenging one due to limited availability of suitable specimen-preparation techniques and narrow applicability of conventional imaging techniques that require at least 2-dimensional crystals for high resolution analysis by electron microscopy (EM) and X-ray diffraction [1][2][3][4][5][6][7][8][9][10][11]. Atomic force microscopy (AFM), which does not require crystallization of the samples for imaging, is emerging as a powerful tool for analyzing biological samples at molecular resolutions [12,13].…”

Location and properties of the digitalis receptor site in Na+/K+‐ATPase

“…In the ~z-fl assembly, the aminoacyl residues between membrane domains H7 and H8 (possibly also from the H9-H10 domains in the carboxyl-terminal portion of c~-subunit are involved [26], while in the c~ dimer formation the transmembrane segments in the aminoterminal portion of ~-subunit are responsible [27]. Since the segments H1 and H2 are associated in pairs [28], the interface in the dimer could be formed by four transmembrane segments.…”

Cryo-electron microscope analysis of frozen-hydrated crystals of Na, K-ATPase.

“…In the dimeric crystals the two protomers have been observed in projection images to be similar (4) or dissimilar (2). Also three-dimensional reconstructions of the Na,K-ATPase protein in dimeric crystals have shown unit cells with either two identical protomers (8,18,20) or two differently shaped protomers in each unit cell (6). Recent cryo-electron microscopy of unfixed and unstained two-dimensional cryostals of Na,KATPase in frozen-hydrated preparations have confirmed that the projection structure of the enzyme protein in the crystalline arrays are either dimeric (16,17) or monomeric (16).…”