Isoform specificity of ankyrin-B - A site in the divergent C-terminal domain is required for intramolecular association
Isoform specificity of ankyrin-B - A site in the divergent C-terminal domain is required for intramolecular association
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DOI：
10.1074/jbc.m506697200
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发表时间：
2006-03-03
影响因子：
4.8
通讯作者：
Bennett, V
中科院分区：
文献类型：
--
作者：
Abdi, KM;Mohler, PJ;Bennett, V
锚蛋白含有显著的氨基酸同一性，并且在许多细胞类型中共表达，但在体内保持独特的功能。最近的研究已经确定了高度分歧的C-末端结构域在心肌细胞中的关键结构域驱动的ankrex-B的特异性功能。在这里，我们确定了分子内相互作用的C-末端结构域和膜结合结构域的锚蛋白- B在溶液中使用纯蛋白和酵母双杂交测定。通过广泛的删除和丙氨酸扫描诱变，我们已经映射在这两个领域的相互作用的关键残基。位于锚蛋白- B C-末端结构域的氨基酸（EED 1599）-E-1597和位于ANK重复序列1的氨基酸Arg（37）/Arg（40）对于酵母双杂交测定中的结构域间相互作用是必需的。此外，氨基酸EED 1597转化为AAA（1597）导致锚蛋白- B突变心肌细胞中肌醇1，4，5-三磷酸受体定位功能丧失。通过圆二色性光谱和流体动力学参数确定的锚蛋白- B C-末端结构域的物理性质揭示了它是非结构化的并且在溶液中高度延伸。对全长220-kDa锚蛋白- B进行的类似结构研究（1597）AAA（1599）显示，与野生型锚蛋白- B相比，其构象更长。总之，这些结果表明一个模型的扩展和非结构化的C-末端结构域折叠回绑定和潜在的调节膜结合结构域的锚蛋白- B。
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 (EED1599)-E-1597 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 EED1597 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.