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

Published

Journal Article

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 (1597)EED(1599) 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 EED(1597) 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.

Full Text

Duke Authors

Cited Authors

  • Abdi, KM; Mohler, PJ; Davis, JQ; Bennett, V

Published Date

  • March 3, 2006

Published In

Volume / Issue

  • 281 / 9

Start / End Page

  • 5741 - 5749

PubMed ID

  • 16368689

Pubmed Central ID

  • 16368689

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.M506697200

Language

  • eng

Conference Location

  • United States