Goode B L, Denis P E, Panda D, Radeke M J, Miller H P, Wilson L, Feinstein S C
Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara 93106, USA.
Mol Biol Cell. 1997 Feb;8(2):353-65. doi: 10.1091/mbc.8.2.353.
Tau is a neuronal microtubule-associated protein that promotes microtubule assembly, stability, and bundling in axons. Two distinct regions of tau are important for the tau-microtubule interaction, a relatively well-characterized "repeat region" in the carboxyl terminus (containing either three or four imperfect 18-amino acid repeats separated by 13- or 14-amino acid long inter-repeats) and a more centrally located, relatively poorly characterized proline-rich region. By using amino-terminal truncation analyses of tau, we have localized the microtubule binding activity of the proline-rich region to Lys215-Asn246 and identified a small sequence within this region, 215KKVAVVR221, that exerts a strong influence on microtubule binding and assembly in both three- and four-repeat tau isoforms. Site-directed mutagenesis experiments indicate that these capabilities are derived largely from Lys215/Lys216 and Arg221. In marked contrast to synthetic peptides corresponding to the repeat region, peptides corresponding to Lys215-Asn246 and Lys215-Thr222 alone possess little or no ability to promote microtubule assembly, and the peptide Lys215-Thr222 does not effectively suppress in vitro microtubule dynamics. However, combining the proline-rich region sequences (Lys215-Asn246) with their adjacent repeat region sequences within a single peptide (Lys215-Lys272) enhances microtubule assembly by 10-fold, suggesting intramolecular interactions between the proline-rich and repeat regions. Structural complexity in this region of tau also is suggested by sequential amino-terminal deletions through the proline-rich and repeat regions, which reveal an unusual pattern of loss and gain of function. Thus, these data lead to a model in which efficient microtubule binding and assembly activities by tau require intramolecular interactions between its repeat and proline-rich regions. This model, invoking structural complexity for the microtubule-bound conformation of tau, is fundamentally different from previous models of tau structure and function, which viewed tau as a simple linear array of independently acting tubulin-binding sites.
Tau是一种神经元微管相关蛋白，可促进轴突中微管的组装、稳定和束集。Tau的两个不同区域对Tau-微管相互作用很重要，一个是位于羧基末端特征相对明确的“重复区域”（包含三个或四个不完美的18个氨基酸重复序列，由13或14个氨基酸长的重复间隔序列隔开），另一个是位于更中心位置、特征相对不明确的富含脯氨酸的区域。通过对Tau进行氨基末端截短分析，我们已将富含脯氨酸区域的微管结合活性定位到Lys215-Asn246，并在该区域内鉴定出一个小序列215KKVAVVR221，该序列对三重复和四重复Tau异构体中的微管结合和组装有强烈影响。定点诱变实验表明，这些能力很大程度上源自Lys215/Lys216和Arg221。与对应于重复区域的合成肽形成鲜明对比的是，单独对应于Lys211515-Asn246和Lys215-Thr222的肽几乎没有促进微管组装的能力，并且肽Lys215-Thr222不能有效抑制体外微管动力学。然而，将富含脯氨酸的区域序列（Lys215-Asn246）与其在单个肽（Lys215-Lys272）中的相邻重复区域序列相结合，可使微管组装增强10倍，这表明富含脯氨酸区域和重复区域之间存在分子内相互作用。通过富含脯氨酸区域和重复区域进行连续的氨基末端缺失也表明了Tau该区域的结构复杂性，这揭示了一种不寻常的功能丧失和获得模式。因此，这些数据得出一个模型，其中Tau有效的微管结合和组装活性需要其重复区域和富含脯氨酸区域之间的分子内相互作用。该模型认为Tau与微管结合的构象具有结构复杂性，这与之前将Tau视为由独立作用的微管蛋白结合位点组成的简单线性阵列的Tau结构和功能模型有根本不同。
