NUCLEAR-MAGNETIC-RESONANCE CHARACTERIZATION OF THE JUN LEUCINE-ZIPPER DOMAIN - UNUSUAL PROPERTIES OF COILED-COIL INTERFACIAL POLAR RESIDUES
NUCLEAR-MAGNETIC-RESONANCE CHARACTERIZATION OF THE JUN LEUCINE-ZIPPER DOMAIN - UNUSUAL PROPERTIES OF COILED-COIL INTERFACIAL POLAR RESIDUES
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DOI：
10.1021/bi00018a020
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发表时间：
1995-05-09
期刊：
影响因子：
2.9
通讯作者：
KING, GF
中科院分区：
文献类型：
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作者：
JUNIUS, FK;MACKAY, JP;KING, GF
亮氨酸拉链构成了广泛观察到的结构基序，其有助于促进许多 DNA 结合蛋白的同二聚化和异二聚化。作为我们持续努力表征该二聚化结构域与生物功能相关的结构和动力学特性的一部分，我们在此报告了重组二聚肽 (rJunLZ) 溶液中的二级结构，该重组二聚肽 (rJunLZ) 包含 c-Jun 亮氨酸拉链结构域的残基 Arg276-Asn314。二维和三维同核和异核核磁共振实验已经允许定义rJunLZ的二级结构，并为三级结构计算提供了总共大约1500个质子间距离和62 phi二面角约束。根据氢-氘交换实验计算得出的酰胺质子保护因子在 rJunLZ 二聚体中发现了 62 个氢键。我们还研究了 Asn22 的作用，Asn22 是位于疏水二聚体界面的唯一极性残基。事实上，所有亮氨酸拉链序列在基序中心附近都含有这样的极性残基（通常是天冬氨酸）。 X 射线晶体学研究表明，就 GCN4 同二聚体而言，极性残基 (Asn) 在基本对称的结构中采用不对称构象。相比之下，迄今为止所有亮氨酸拉链同型二聚体的 NMR 研究表明，二聚体在溶液中是完全对称的。我们提供的证据表明，Asn22 的侧链酰胺质子在溶液中形成氢键，并且该侧链在两种不同构象之间快速交换。基于这些观察，我们提出了一个动态模型，可以解释在亮氨酸拉链同二聚体的 NMR 和 X 射线晶体学研究中观察到的对称性的明显差异。我们发现，Asn22 突变为疏水性 Leu 残基可显着提高 rJunLZ 同二聚体的热稳定性，这与该残基的不稳定作用一致。然而，在低于 30 摄氏度的温度下，Asn22 --> Leu 突变体重新排列，形成比二聚体更大的寡聚体，正如之前在 GCN4 亮氨酸拉链中相应的 Asn --> Val 突变所观察到的那样。这些结果与以下假设一致：亮氨酸拉链界面处常见的极性 Asn 残基以牺牲稳定性为代价强加了二聚体结构的特异性 [Harbury, P. B.、Zhang, T.、Kim, P. S. 和 Alber, T. (1993) Science 262, 1401-1407]。
Leucine zippers constitute a widely observed structural motif which serves to promote both homo- and heterodimerization in a number of DNA-binding proteins. As part of our ongoing efforts to characterize both the structure and the dynamical properties of this dimerization domain as they relate to biological function, we report here the secondary structure in solution of a recombinant dimeric peptide (rJunLZ) comprising residues Arg276-Asn314 of the leucine zipper domain of c-Jun. Two- and three-dimensional homo- and heteronuclear NMR experiments have allowed definition of the secondary structure of rJunLZ and have provided a total of similar to 1500 interproton distance and 62 phi dihedral angle constraints for tertiary structure calculations. Amide proton protection factors, calculated from hydrogen-deuterium exchange experiments, have identified 62 hydrogen bonds in the rJunLZ dimer. We have also examined the role of Asn22, the only polar residue situated at the hydrophobic dimer interface. Virtually all leucine zipper sequences contain such a polar residue (usually Asn) near the center of the motif. X-ray crystallographic studies showed that, in the case of the GCN4 homodimer, the polar residue (Asn) adopts an asymmetric conformation in an otherwise essentially symmetric structure. In contrast, all NMR studies of leucine zipper homodimers to date have suggested that the dimers are completely symmetric in solution. We present evidence that the side-chain amide protons of Asn22 are hydrogen-bonded in solution and that this side chain exchanges rapidly between two distinct conformations. On the basis of these observations, we propose a dynamic model which can explain the apparent differences in symmetry observed in NMR and X-ray crystallographic studies of leucine zipper homodimers. We show that mutation of Asn22 to a hydrophobic Leu residue markedly increases the thermal stability of the rJunLZ homodimer, consistent with a destabilizing role for this residue. However, at temperatures below 30 degrees C, the Asn22 --> Leu mutant rearranges to form oligomers larger than the dimer, as was previously observed for the corresponding Asn --> Val mutation in the GCN4 leucine zipper. These results are consistent with the hypothesis that the polar Asn residue commonly observed at the interface of leucine zippers imposes specificity for the dimer structure at the expense of stability [Harbury, P. B., Zhang, T., Kim, P. S., and Alber, T. (1993) Science 262, 1401-1407].