A model for the homotypic interaction between Na⁺,K⁺-ATPase β1 subunits reveals the role of extracellular residues 221-229 in its ig-like domain

The Na⁺, K⁺-ATPase transports Na⁺ and K⁺ across the membrane of all animal cells. In addition to its ion transporting function, the Na⁺, K⁺-ATPase acts as a homotypic epithelial cell adhesion molecule via its β1 subunit. The extracellular region of the Na⁺, K⁺-ATPase β1 subunit includes a single globular immunoglobulin-like domain. We performed Molecular Dynamics simulations of the ectodomain of the β1 subunit and a refined protein-protein docking prediction. Our results show that the β1 subunit Ig-like domain maintains an independent structure and dimerizes in an antiparallel fashion. Analysis of the putative interface identified segment Lys221-Tyr229. We generated triple mutations on YFP-β1 subunit fusion proteins to assess the contribution of these residues. CHO fibroblasts transfected with mutant β1 subunits showed a significantly decreased cell-cell adhesion. Association of β1 subunits in vitro was also reduced, as determined by pull-down assays. Altogether, we conclude that two Na⁺, K⁺-ATPase molecules recognize each other by a large interface spanning residues 221-229 and 198-207 on their β1 subunits.