Structural studies on a protein-binding zinc-finger domain of eos reveal both similarities and differences to classical zinc fingers
Structural studies on a protein-binding zinc-finger domain of eos reveal both similarities and differences to classical zinc fingers
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DOI：
10.1021/bi049506a
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发表时间：
2004-10-26
期刊：
影响因子：
2.9
通讯作者：
Mackay, JP
中科院分区：
文献类型：
--
作者：
Westman, BJ;Perdomo, J;Mackay, JP
存在于Ikaros家族蛋白和蛋白Trps-1的C末端的寡聚化结构域对于发育过程如造血的适当调节是重要的。值得注意的是，该结构域被预测含有两个经典的锌指（ZnFs），通常与核酸识别相关的结构域。这些预测的ZnFs对蛋白质结合的偏好还没有得到很好的理解。我们已经使用了一系列的方法来深入了解这个域的结构。对Eos的C-末端结构域（EosC）进行的圆二色性、UV-vis和NMR实验揭示了两种推定的ZnF（C1和C2）是可分离的，即，在Zn-II存在下能够独立折叠。接下来，我们使用NMR光谱确定了EosC 2的结构，揭示了尽管EosC 2的整体折叠与其他经典的ZnFs相似，但存在许多差异。例如，EosC 2的C末端的构象似乎是灵活的，并且可能导致锌配体的主要重排。最后，丙氨酸扫描诱变被用来确定参与的同源和异源寡聚化的EOS的残基，这些结果进行了讨论的背景下的EosC的结构。这些研究为EosC如何介导蛋白质-蛋白质相互作用提供了第一个结构性见解，并有助于我们理解为什么它不表现出高亲和力DNA结合。
The oligomerization domain that is present at the C terminus of Ikaros-family proteins and the protein Trps-1 is important for the proper regulation of developmental processes such as hematopoiesis. Remarkably, this domain is predicted to contain two classical zinc fingers (ZnFs), domains normally associated with the recognition of nucleic acids. The preference for protein binding by these predicted ZnFs is not well-understood. We have used a range of methods to gain insight into the structure of this domain. Circular dichroism, UV-vis, and NMR experiments carried out on the C-terminal domain of Eos (EosC) revealed that the two putative ZnFs (C1 and C2) are separable, i.e., capable of folding independently in the presence of Zn-II. We next determined the structure of EosC2 using NMR spectroscopy, revealing that, although the overall fold of EosC2 is similar to other classical ZnFs, a number of differences exist. For example, the conformation of the C terminus of EosC2 appears to be flexible and may result in a major rearrangement of the zinc ligands. Finally, alanine-scanning mutagenesis was used to identify the residues that are involved in the homo- and hetero-oligomerization of Eos, and these results are discussed in the context of the structure of EosC. These studies provide the first structural insights into how EosC mediates protein-protein interactions and contributes to our understanding of why it does not exhibit high-affinity DNA binding.