Covalent cross-links between the gamma subunit (FXYD2) and alpha and beta subunits of Na,K-ATPase: modeling the alpha-gamma interaction.
This study describes specific intramolecular covalent cross-linking of the gamma to alpha and gamma to beta subunits of pig kidney Na,K-ATPase and rat gamma to alpha co-expressed in HeLa cells. For this purpose pig gammaa and gammab sequences were determined by cloning and mass spectrometry. Three bifunctional reagents were used: N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA), disuccinimidyl tartrate (DST), and 1-ethyl-3-[3dimethylaminopropyl]carbodiimide (EDC). NHS-ASA induced alpha-gamma, DST induced alpha-gamma and beta-gamma, and EDC induced primarily beta-gamma cross-links. Specific proteolytic and Fe(2+)-catalyzed cleavages located NHS-ASA- and DST-induced alpha-gamma cross-links on the cytoplasmic surface of the alpha subunit, downstream of His(283) and upstream of Val(440). Additional considerations indicated that the DST-induced and NHS-ASA-induced cross-links involve either Lys(347) or Lys(352) in the S4 stalk segment. Mutational analysis of the rat gamma subunit expressed in HeLa cells showed that the DST-induced cross-link involves Lys(55) and Lys(56) in the cytoplasmic segment. DST and EDC induced two beta-gamma cross-links, a major one at the extracellular surface within the segment Gly(143)-Ser(302) of the beta subunit and another within Ala(1)-Arg(142). Based on the cross-linking and other data on alpha-gamma proximities, we modeled interactions of the transmembrane alpha-helix and an unstructured cytoplasmic segment SKRLRCGGKKHR of gamma with a homology model of the pig alpha1 subunit. According to the model, the transmembrane segment fits in a groove between M2, M6, and M9, and the cytoplasmic segment interacts with loops L6/7 and L8/9 and stalk S5.
Füzesi
Maria
M
Department of Biological Chemistry and Biological Mass Spectrometry Facility, Weizmann Institute of Science, Rehovoth, 76100, Israel.
Gottschalk
Kay-Eberhard
KE
Lindzen
Moshit
M
Shainskaya
Alla
A
Küster
Bernhard
B
Garty
Haim
H
Karlish
Steven J D
SJ
D000595:Amino Acid Sequence; D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004252:DNA Mutational Analysis; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping / Q000379:methods*; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013552:Swine
D003432:Cross-Linking Reagents; D021122:Protein Subunits; C416502:FXYD2 protein, human; C498817:Fxyd2 protein, rat; D000254:Sodium-Potassium-Exchanging ATPase