Thermal Denaturation of the Na,K-ATPase Provides Evidence for α-α Oligomeric Interaction and γ Subunit Association with the C-terminal Domain

Abstract

Thermal denaturation can help elucidate protein domain substructure. We previously showed that the Na,K-ATPase partially unfolded when heated to 55°C (Arystarkhova, E., Gibbons, D. L., and Sweadner, K. J. (1995) J. Biol. Chem. 270, 8785-8796). The β subunit unfolded without leaving the membrane, but three transmembrane spans (M8-M10) and the C terminus of the α subunit were extruded, while the rest of α retained its normal topology with respect to the lipid bilayer. Here we investigated thermal denaturation further, with several salient results. First, trypsin sensitivity at both surfaces of α was increased, but not sensitivity to V8 protease, suggesting that the cytoplasmic domains and extruded domain were less tightly packed but still retained secondary structure. Second, thermal denaturation was accompanied by SDS-resistant aggregation of α subunits as dimers, trimers, and tetramers without β or γ subunits. This implies specific α-α contact. Third, the γ subunit, like the C-terminal spans of α, was selectively lost from the membrane. This suggests its association with M8-M10 rather than the more firmly anchored transmembrane spans. The picture that emerges is of a Na,K-ATPase complex of α, β, and γ subunits in which α can associate in assemblies as large as tetramers via its cytoplasmic domain, while β and γ subunits associate with α primarily in its C-terminal portion, which has a unique structure and thermal instability.