Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232-0615, USA.
J Biol Chem. 2011 Jun 10;286(23):20746-57. doi: 10.1074/jbc.M111.230326. Epub 2011 Apr 14.
The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18-20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.
衔接蛋白 ankyrin-R 通过其膜结合域与阴离子交换蛋白 (AE1) 的细胞质域相互作用，并通过其 spectrin 结合域与人类红细胞中的 spectrin 基底膜骨架相互作用。这组相互作用在脂质双层和膜骨架之间提供了桥梁，从而稳定了膜。已经报道了 AE1 的二聚体细胞质结构域 (cdb3) 和来自 ankyrin-R 的膜结合结构域 (AnkD34) 的 12-ankyrin 重复片段 (重复 13-24) 的晶体结构。然而，关于这些蛋白质如何组装形成稳定复合物的结构数据尚未报道。在当前的研究中，已利用定点旋转标记，结合电子顺磁共振 (EPR) 和双电子电子共振，来绘制复合物中两种蛋白质的结合界面，并获得蛋白质间的距离约束。这些数据已用于构建一系列结构模型，这些模型与全范围的实验数据一致。这些模型表明，cdb3 的外周结构域的一个广泛区域主要通过疏水相互作用与 AnkD34 顶部环表面的重复 18-20 结合。这是 cdb3 与 AnkD34 结合的一个以前未表征的表面。因为已知第二个 cdb3 二聚体与 ankyrin-R 的膜结合结构域的重复 7-12 结合，当前的模型对于假定参与与红细胞膜中全长 ankyrin-R 结合的 AE1 四聚体形式的结构性质具有重要意义。
