Isoform specificity among ankyrins. An amphipathic alpha-helix in the divergent regulatory domain of ankyrin-b interacts with the molecular co-chaperone Hdj1/Hsp40.
Ankyrins-R, -B, and -G are a family of membrane-associated adaptors required for localization of structurally diverse proteins to specialized membrane domains, including axon initial segments, cardiomyocyte T-tubules, and epithelial cell lateral membranes. Ankyrins are often co-expressed in the same cells and, although structurally similar, have non-overlapping functions. We previously determined that the regulatory domain of ankyrin-B defines specificity between ankyrins B and G in cardiomyocytes. Here, we identify key residues on the surface of an amphipathic alpha-helix unique to the regulatory domain of ankyrin-B that are essential for the function of ankyrin-B in cardiomyocytes. Using circular dichroism, we determined that a peptide representing the predicted helix folds as a helix in solution. Alanine-scanning mutagenesis revealed that residues 1773, 1777, 1780, 1784, and 1788 located in a patch on one surface the helix are critical for ankyrin-B function in cardiomyocytes. In a parallel set of experiments we determined that the molecular co-chaperone human DnaJ homologue 1 (Hdj1)/Hsp40 interacts with the ankyrin-B regulatory domain. Moreover, interaction of Hdj1/Hsp40 with the regulatory domain was mapped by random mutagenesis to same surface of the alpha-helix that is required for ankyrin-B function. These results provide new insight into the molecular basis for specificity between ankyrin-based pathways by defining a key alpha-helix structure in the divergent regulatory domain of ankyrin-B as well as interaction of the helix with Hdj1/Hsp40, the first downstream target for ankyrin-B-specific function.
Mohler
Peter J
PJ
Howard Hughes Medical Institute and Departments of Cell Biology, Biochemistry, and Neurosciences, Duke University Medical Center, Durham, North Carolina 27710, USA. pmohler@cellbio.duke.edu
Hoffman
Janis A
JA
Davis
Jonathan Q
JQ
Abdi
Khadar M
KM
Kim
Chong-Rak
CR
Jones
Sarah K
SK
Davis
Lydia H
LH
Roberts
Karen F
KF
Bennett
Vann
V
D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000737:chemistry* / Q000502:physiology; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000502:physiology; D020033:Protein Isoforms; D017433:Protein Structure, Secondary
C498994:ANK2 protein, human; C498995:Ank2 protein, mouse; D017487:Ankyrins; C494647:DNAJB1 protein, human; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D020033:Protein Isoforms