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 (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.
Abdi
Khadar M
KM
Howard Hughes Medical Institute, and Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.
Mohler
Peter J
PJ
Davis
Jonathan Q
JQ
Bennett
Vann
V
D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques
D017487:Ankyrins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins