Oligomerization of the polycystin-2 C-terminal tail and effects on its Ca2+-binding properties.
Oligomerization of the polycystin-2 C-terminal tail and effects on its Ca2+-binding properties.
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DOI：
10.1074/jbc.m115.641803
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发表时间：
2015-04-17
期刊：
影响因子：
--
通讯作者：
Hodsdon ME
中科院分区：
文献类型：
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作者：
Yang Y;Keeler C;Kuo IY;Lolis EJ;Ehrlich BE;Hodsdon ME
背景：多囊蛋白-2（PC 2 Cterm）的C末端尾对于通道组装和调节是必需的。结果：人和海胆PC 2 Cterm均形成三聚体，并含有与Ca 2+结合的EF-手结构域。结论：寡聚化对人和海胆PC 2中的Ca 2+结合谱的影响不同。意义：PC 2 Cterm的表征有助于理解PC 2通道调节。多囊蛋白-2（PC 2）属于瞬时受体电位（TRP）家族，形成钙离子调节通道。人PC 2的C-末端胞质尾区（HPC 2 Cterm）对于PC 2通道组装和调节是重要的。在这项研究中，我们的特点是寡聚状态和Ca 2+结合的C-末端尾巴使用生物物理方法。具体地说，我们确定了HPC 2 Cterm在有和没有Ca 2+结合的溶液中形成三聚体，尽管TRP通道被认为是四聚体。我们发现在HPC 2 Cterm中只有一个Ca ~（2+）结合位点，位于其EF-手结构域内。然而，相对于分离的EF-手结构域的固有结合亲和力，HPC 2 Cterm三聚体的Ca 2+结合亲和力大大增强。我们还采用海胆PC 2（SUPC 2）作为生物物理和结构表征的模型。海胆C-末端结构（SUPC 2 Ccore）也在溶液中形成三聚体，不依赖于Ca 2+结合。与人PC 2相反，SUPC 2 Ccore在其EF-手结构域内包含两个协同的Ca 2+结合位点。因此，相对于分离的EF-手结构域，三聚化不会进一步提高SUPC 2 C核心中的Ca 2+结合的亲和力。使用NMR，我们本地化的SUPC 2 C核心的钙离子结合位点，其特征在于在其EF-手域由于三聚体形成的构象变化。我们的研究提供了一个结构上的基础，了解钙离子依赖性调节的PC 2通道的胞质C-末端结构域。改进的方法也可以作为一个很好的策略，以表征其他Ca 2+结合蛋白。
Background: The C-terminal tail of polycystin-2 (PC2 Cterm) is essential for channel assembly and regulation. Results: Both human and sea urchin PC2 Cterm form trimers and contain EF-hand domains that bind to Ca2+. Conclusion: Oligomerization affects Ca2+-binding profiles differently in human and sea urchin PC2. Significance: Characterization of the PC2 Cterm aids the understanding of PC2 channel regulation. Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca2+-regulated channel. The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is important for PC2 channel assembly and regulation. In this study, we characterized the oligomeric states and Ca2+-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca2+ bound, although TRP channels are believed to be tetramers. We found that there is only one Ca2+-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca2+ binding affinity of the HPC2 Cterm trimer is greatly enhanced relative to the intrinsic binding affinity of the isolated EF-hand domain. We also employed the sea urchin PC2 (SUPC2) as a model for biophysical and structural characterization. The sea urchin C-terminal construct (SUPC2 Ccore) also forms trimers in solution, independent of Ca2+ binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca2+-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca2+ binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca2+-binding sites in the SUPC2 Ccore and characterized the conformational changes in its EF-hand domain due to trimer formation. Our study provides a structural basis for understanding the Ca2+-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca2+-binding proteins.