A highly conserved region of human erythrocyte ankyrin contains the capacity to bind spectrin.
Ankyrin has a spectrin-binding region within a central 62-kDa chymotryptic peptide. We examined the spectrin binding ability of a series of smaller ankyrin fragments and recombinant peptides within the 62-kDa domain using a ligand blot assay. The smallest proteolytic fragment that bound was a 12-kDa tryptic peptide starting at amino acid 1068. Peptides containing this region expressed as glutathione S-transferase fusion products also bound spectrin and suggested that residues 1101-1192 were important. In contrast, a fusion protein containing residues 826-898 did not bind spectrin, a surprising finding since this region is known to influence binding affinity. Proteins that bound spectrin on ligand blots also competed for binding in solution, but did so with one-tenth the affinity of the native peptide. Comparing the 62-kDa domains of erythrocyte and brain ankyrins (species that bind spectrin but with 10-fold differences in affinity), the NH2-terminal regions are 0-40% identical, while the regions (1136-1160) common to all binding peptides are 80-90% identical. We hypothesize that the highly conserved region contains an important spectrin-binding site, while the poorly conserved region controls the binding affinity. We speculate that this unique NH2-terminal region is what gives different members of the ankyrin family their signature set of affinities, and accordingly their distinctive cellular localization.
Platt
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Department of Medicine, Children's Hospital, Boston, Massachusetts.
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D000595:Amino Acid Sequence; D017487:Ankyrins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D017931:DNA Primers; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013049:Spectrin / Q000378:metabolism*
D017487:Ankyrins; D017931:DNA Primers; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013049:Spectrin