Covalent cross-links between the γ subunit (FXYD2) and α and β subunits of Na,K-ATPase: Modeling the α-γ interaction

This study describes specific intramolecular covalent cross-linking of the γ to α and γ to β subunits of pig kidney Na,K-ATPase and rat γ to α co-expressed in HeLa cells. For this purpose pig -γa and -γb 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 α-γ, DST induced α-γ and β-γ, and EDC induced primarily β-γ cross-links. Specific proteolytic and Fe ²⁺-catalyzed cleavages located NHS-ASA- and DST-induced α-γ cross-links on the cytoplasmic surface of the α subunit, downstream of His²⁸³ and upstream of Val⁴⁴⁰. Additional considerations indicated that the DST-induced and NHS-ASA-induced cross-links involve either Lys³⁴⁷ or Lys³⁵² in the S4 stalk segment. Mutational analysis of the rat γ subunit expressed in HeLa cells showed that the DST-induced cross-link involves Lys⁵⁵ and Lys⁵⁶ in the cytoplasmic segment. DST and EDC induced two β-γ cross-links, a major one at the extracellular surface within the segment Gly ¹⁴³-Ser³⁰² of the β subunit and another within Ala¹-Arg¹⁴². Based on the cross-linking and other data on α-γ proximities, we modeled interactions of the transmembrane α-helix and an unstructured cytoplasmic segment SKRLRCG-GKKHR of γ with a homology model of the pig α1 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.