Isoform specificity of ankyrin-B - A site in the divergent C-terminal domain is required for intramolecular association

Ankyrins contain significant amino acid identity and are co-expressed in many cell types yet maintain unique functions in vivo. Recent studies have identified the highly divergent C-terminal domain in ankyrin-B as the key domain for driving ankyrin-B-specific functions in cardiomyocytes. Here we identify an intramolecular interaction between the C-terminal domain and the membrane-binding domain of ankyrin- B using pure proteins in solution and the yeast two-hybrid assay. Through extensive deletion and alanine-scanning mutagenesis we have mapped key residues for interaction in both domains. Amino acids (EED1599)-E-1597 located in the ankyrin- B C-terminal domain and amino acids Arg(37)/Arg(40) located in ANK repeat 1 are necessary for inter-domain interactions in yeast two-hybrid assays. Furthermore, conversion of amino acids EED1597 to AAA(1597) leads to a loss of function in the localization of inositol 1,4,5-trisphosphate receptors in ankyrin- B mutant cardiomyocytes. Physical properties of the ankyrin- B C-terminal domain determined by circular dichroism spectroscopy and hydrodynamic parameters reveal it is unstructured and highly extended in solution. Similar structural studies performed on full-length 220-kDa ankyrin- B harboring alanine substitutions, (1597)AAA(1599), reveal a more extended conformation compared with wild-type ankyrin- B. Taken together these results suggest a model of an extended and unstructured C-terminal domain folding back to bind and potentially regulate the membrane-binding domain of ankyrin- B.