Packing of the transmembrane helices of Na,K-ATPase: Direct contact between beta-subunit and H8 segment of alpha-subunit revealed by oxidative cross-linking

Citation
A. Ivanov et al., Packing of the transmembrane helices of Na,K-ATPase: Direct contact between beta-subunit and H8 segment of alpha-subunit revealed by oxidative cross-linking, BIOCHEM, 39(32), 2000, pp. 9778-9785
Citations number
45
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
0006-2960 → ACNP
Volume
39
Issue
32
Year of publication
2000
Pages
9778 - 9785
Database
ISI
SICI code
0006-2960(20000815)39:32<9778:POTTHO>2.0.ZU;2-M
Abstract
Spatial relationships among the transmembrane (TM) segments of alpha- and b eta-subunits of the Na,K-ATPase molecule have been investigated using oxida tive induction of disulfide bonds. The catalytic cr-subunit contains 10 TM alpha-helices (H1-H10) with 9 Cys residues located within or close to the m embrane moiety. There is one Cys residue in the single TM segment of beta-s ubunit (H beta). Previously, the crosslinking products containing the beta- subunit and two fragments of alpha-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 o f the membrane moiety of trypsin-digested Na,K-ATPase [Sarvazyan, N. A., Mo dyanov, N. N., and Askari, A. (1995) J. Biol. Chem. 270, 26528-26532 and Sa rvazyan, N. A., Ivanov, A., Modyanovl N. N., and Askari, A. (1997) J. Biol. Chem. 272, 7855-7858]. Here, we have shown that Cu2+-phenanthroline treatm ent of digitonin-solubilized preparation provides the most efficient format ion of intersubunit cross-linked product that is predominantly a dimer of b eta-subunit and a 22-kDa C-terminal alpha-fragment containing H7 -H10 helic es. 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 ne853-Met932 alpha-fragment and AIa5-Met56 beta -fragment was identified as a product of intersubunit disulfide cross-link between Cys44 of H beta and either Cys911 or Cys930, located in H8. This pr ovides the first direct experimental evidence of the juxtaposition of HP an d H8 within the Na,K-ATPase molecule. The second detected cross-linked prod uct was composed of ct-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 H 10 helices of alpha-subunit.