Ding Y, Kobayashi S, Kopito R
Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA.
J Biol Chem. 1996 Sep 13;271(37):22494-8. doi: 10.1074/jbc.271.37.22494.
The association of ankyrin with the AE1 anion exchanger contributes an essential function to the mechanical and viscoelastic properties of the erythrocyte and constitutes the best understood link between the plasma membrane and the underlying membrane skeleton. The AE1 binding domain of ankyrin consists of 24 tandem repeats of a 33-amino acid motif that is present on a wide variety of otherwise unrelated proteins. The experiments described in this paper are aimed at identifying the specific amino acid sequences in AE1 that comprise the ankyrin binding site. We have exploited a cell-free binding assay to quantify the binding affinity of anion exchangers and a recombinant fragment of ANK1, R13-H. Our previous study (Ding, Y., Casey, J. R. and Kopito, R. R. (1995) J. Biol. Chem. 269, 32201-32208) identified an essential role of the amino-terminal 79 AE1 residues in ankyrin binding. The present study extends these findings to show that these 79 amino acids, although necessary, are not sufficient for ankyrin binding. Using chimeras between AE1 and the closely related anion exchanger AE2, which does not bind ankyrin, we have defined a 40-residue region of AE1 between positions 155 and 195 that is also essential for ankyrin binding.
锚蛋白与AE1阴离子交换蛋白的结合对红细胞的机械和粘弹性特性起着至关重要的作用，并且构成了质膜与下方膜骨架之间最易理解的联系。锚蛋白的AE1结合结构域由一个33个氨基酸基序的24个串联重复序列组成，该基序存在于多种其他不相关的蛋白质上。本文所述的实验旨在确定AE1中构成锚蛋白结合位点的特定氨基酸序列。我们利用无细胞结合试验来量化阴离子交换蛋白与ANK1的重组片段R13-H的结合亲和力。我们之前的研究（丁，Y.，凯西，J.R.和科皮托，R.R.（1995年）《生物化学杂志》269，32201 - 32208）确定了AE1氨基末端79个残基在锚蛋白结合中的关键作用。本研究扩展了这些发现，表明这79个氨基酸虽然是必需的，但对于锚蛋白结合并不充分。通过使用AE1与不结合锚蛋白的密切相关阴离子交换蛋白AE2之间的嵌合体，我们确定了AE1在155至195位之间的一个40个残基的区域，该区域对于锚蛋白结合也是必不可少的。
