Davis L H, Otto E, Bennett V
Howard Hughes Medical Institute, Duke University Medical Center, Durham North Carolina 27710.
J Biol Chem. 1991 Jun 15;266(17):11163-9.
Erythrocyte ankyrin contains an 89-kDa domain (residues 2-827) comprised almost entirely of 22 tandem repeats of 33 amino acids which are responsible for the high affinity interaction of ankyrin with the anion exchanger (Davis, L., and Bennett, V. (1990) J. Biol. Chem. 265, 10589-10596). The question of whether the repeats are equivalent with respect to binding to the anion exchanger was addressed using defined regions of erythrocyte and brain ankyrins expressed in bacteria. The conclusion is that the repeats are not interchangeable and that the 44 residues from 722 to 765 are essential for high affinity binding between erythrocyte ankyrin and the anion exchanger. Residues 348-765 were active whereas a polypeptide of the same size (residues 305-721) but missing the 44 residues was not active. The difference between the active and inactive polypeptides was not caused by the degree of folding based on circular dichroism spectra. The 44 residues from 722 to 765 were not sufficient for binding since deletions of residues from 348 to 568 resulted in a 10-fold loss of activity. However, the role of residues 348-568 may be at the level of folding rather than a direct contact since the deleted sequences were not active in the absence of 722-765 and since circular dichroism spectra revealed significant loss of structure in the smaller polypeptides. Further evidence that the 33-residue repeats are not equivalent in ability to bind to the anion exchanger is that a region of human brain ankyrin containing 18 33-residue repeats with 67% overall sequence identity to erythrocyte ankyrin was 8-fold less active than a region of erythrocyte ankyrin containing only 12 repeats. The fact that the anion exchanger binds to certain repeats suggests that the other 33-amino acid repeats could interact with proteins distinct from the anion exchanger and provide ankyrin with the potential for considerable diversity in association with membrane proteins as well as cytoplasmic proteins. Tubulin was identified as one example of a protein that can interact with ankyrin repeats that are not recognized by the anion exchanger.
红细胞锚蛋白含有一个89 kDa的结构域（第2至827位氨基酸残基），几乎完全由22个33氨基酸的串联重复序列组成，这些重复序列负责锚蛋白与阴离子交换蛋白的高亲和力相互作用（戴维斯，L.，和贝内特，V.（1990年）《生物化学杂志》265卷，10589 - 10596页）。利用在细菌中表达的红细胞和脑锚蛋白的特定区域，研究了这些重复序列在与阴离子交换蛋白结合方面是否等同的问题。结论是这些重复序列不可互换，并且第722至765位的44个氨基酸残基对于红细胞锚蛋白与阴离子交换蛋白之间的高亲和力结合至关重要。第348 - 765位氨基酸残基具有活性，而相同大小（第305 - 721位氨基酸残基）但缺失这44个氨基酸残基的多肽则无活性。基于圆二色光谱，活性和非活性多肽之间的差异并非由折叠程度引起。第722至765位的44个氨基酸残基不足以实现结合，因为从第348至568位氨基酸残基的缺失导致活性损失了10倍。然而，第348 - 568位氨基酸残基的作用可能在于折叠层面而非直接接触，因为在没有第722 - 765位氨基酸残基的情况下，缺失的序列无活性，并且圆二色光谱显示较小多肽的结构有显著损失。进一步证明33个氨基酸残基的重复序列在与阴离子交换蛋白结合能力上不等同的证据是，人脑海锚蛋白中一个包含18个33氨基酸重复序列且与红细胞锚蛋白总体序列同一性为67%的区域，其活性比红细胞锚蛋白中仅包含12个重复序列的区域低8倍。阴离子交换蛋白与某些重复序列结合这一事实表明，其他33个氨基酸的重复序列可能与不同于阴离子交换蛋白的蛋白质相互作用，并为锚蛋白提供了与膜蛋白以及细胞质蛋白结合时具有相当多样性的潜力。微管蛋白被鉴定为一种能够与阴离子交换蛋白未识别的锚蛋白重复序列相互作用的蛋白质的例子。
