Isoform Specificity of Ankyrin-B A SITE IN THE DIVERGENT C-TERMINAL DOMAIN IS REQUIRED FOR INTRAMOLECULAR ASSOCIATION

Abstract 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 1597EED1599 located in the ankyrin-B C-terminal domain and amino acids Arg37/Arg40 located in ANK repeat 1 are necessary for inter-domain interactions in yeast two-hybrid assays. Furthermore, conversion of amino acids EED1597 to AAA1597 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, 1597AAA1599, 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.