Structure of the ankyrin-binding domain of α-Na,K-ATPase

Abstract

The ankyrin 33-residue repeating motif, an L-shaped structure with protruding β-hairpin tips, mediates specific macromolecular interactions with cytoskeletal, membrane, and regulatory proteins. The association between ankyrin and α-Na,K-ATPase, a ubiquitous membrane protein critical to vectorial transport of ions and nutrients, is required to assemble and stabilize Na,K-ATPase at the plasma membrane. α-Na,K-ATPase binds both red cell ankyrin (Ank(R), a product of the ANK1 gene) and Madin-Darby canine kidney cell ankyrin (Ank(G), a product of the ANK3 gene) utilizing residues 142-166 (SYYQEAKSSKIMESFK NMVPQQALV) in its second cytoplasmic domain. Fusion peptides of glutathione S-transferase incorporating these 25 amino acids bind specifically to purified ankyrin (K(d) = 118 ± 50 nM). The three-dimensional structure (2.6 Å) of this minimal ankyrin-binding motif, crystallized as the fusion protein, reveals a 7-residue loop with one charged hydrophilic face capping a double β-strand. Comparison with ankyrin-binding sequences in p53, CD44, neurofascin/L1, and the inositol 1,4,5-trisphosphate receptor suggests that the valency and specificity of ankyrin binding is achieved by the interaction of 5-7-residue surface loops with the β-hairpin tips of multiple ankyrin repeat units.