Packing of the Transmembrane Helices of Na,K-ATPase:  Direct Contact between β-Subunit and H8 Segment of α-Subunit Revealed by Oxidative Cross-Linking

Spatial relationships among the transmembrane (TM) segments of α- and β-subunits of the Na,K-ATPase molecule have been investigated using oxidative induction of disulfide bonds. The catalytic α-subunit contains 10 TM α-helices (H1−H10) with 9 Cys residues located within or close to the membrane moiety. There is one Cys residue in the single TM segment of β-subunit (Hβ). Previously, the cross-linking products containing the β-subunit and two fragments of α-subunit (the N-terminal containing H1−H2 helices and the C-terminal containing H7−H10 helices) have been identified in experiments with membrane-bound or detergent-solubilized preparations of the membrane moiety of trypsin-digested Na,K-ATPase [Sarvazyan, N. A., Modyanov, N. N., and Askari, A. (1995) J. Biol. Chem. 270, 26528−26532 and Sarvazyan, N. A., Ivanov, A., Modyanov, N. N., and Askari, A. (1997) J. Biol. Chem. 272, 7855−7858]. Here, we have shown that Cu2+-phenanthroline treatment of digitonin-solubilized preparation provides the most efficient formation of intersubunit cross-linked product that is predominantly a dimer of β-subunit and a 22-kDa C-terminal α-fragment containing H7−H10 helices. This cross-linked product was isolated and subjected to CNBr cleavage. The resulting fragments were electrophoretically separated and sequenced. A 17-kDa peptide composed of Ile853−Met942 α-fragment and Ala5−Met56 β-fragment was identified as a product of intersubunit disulfide cross-link between Cys44 of Hβ and either Cys911 or Cys930, located in H8. This provides the first direct experimental evidence of the juxtaposition of Hβ and H8 within the Na,K-ATPase molecule. The second detected cross-linked product was composed of α-fragments Lys947−Met963 and Tyr974−Tyr1016 linked by induced disulfide bridge between Cys964 (H9) and Cys983 (H10). The spatial proximity of these Cys residues defines the mutual orientation of H9 and H10 helices of α-subunit.