Effect of Src Kinase Phosphorylation on Disordered C-terminal Domain of N-Methyl-D-aspartic Acid (NMDA) Receptor Subunit GluN2B Protein
Effect of Src Kinase Phosphorylation on Disordered C-terminal Domain of N-Methyl-D-aspartic Acid (NMDA) Receptor Subunit GluN2B Protein
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DOI：
10.1074/jbc.m111.258897
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发表时间：
2011-08-26
影响因子：
4.8
通讯作者：
Bowen, Mark E.
中科院分区：
文献类型：
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作者：
Choi, Ucheor B.;Xiao, Shifeng;Bowen, Mark E.
NMDA受体是具有调节性细胞内C-末端结构域（CTD）的配体门控离子通道。在GluN 2B中，CTD是蛋白质中最大的结构域，但本质上是无序的。GluN 2B亚基是突触中主要的酪氨酸磷酸化蛋白。Src激酶磷酸化GluN 2B CTD，但尚不清楚这如何影响通道活性。在无序的蛋白质中，磷酸化可以打破有序和无序之间的平衡。转换可以在两个方向上发生，因此目前无法预测磷酸化的影响。我们使用单分子荧光来表征Src磷酸化对GluN 2B的影响。扫描整个域的荧光标记位点显示没有位置依赖性的能量转移。相反，效率仅与标记位点之间的分离成比例，这暗示了相对无特征的构象能量景观。Src磷酸化导致多肽的普遍扩增，这将导致已知蛋白质结合位点的更多暴露，并增加相邻位点之间的物理分离。磷酸化使得CTD更像是一个无规卷曲，留下了Src如何对NMDA受体发挥作用的问题。
NMDA receptors are ligand-gated ion channels with a regulatory intracellular C-terminal domain (CTD). In GluN2B, the CTD is the largest domain in the protein but is intrinsically disordered. The GluN2B subunit is the major tyrosine-phosphorylated protein in synapses. Src kinase phosphorylates the GluN2B CTD, but it is unknown how this affects channel activity. In disordered proteins, phosphorylation can tip the balance between order and disorder. Transitions can occur in both directions, so it is not currently possible to predict the effects of phosphorylation. We used single molecule fluorescence to characterize the effects of Src phosphorylation on GluN2B. Scanning fluorescent labeling sites throughout the domain showed no positional dependence of the energy transfer. Instead, efficiency only scaled with the separation between labeling sites suggestive of a relatively featureless conformational energy landscape. Src phosphorylation led to a general expansion of the polypeptide, which would result in greater exposure of known protein-binding sites and increase the physical separation between contiguous sites. Phosphorylation makes the CTD more like a random coil leaving open the question of how Src exerts its effects on the NMDA receptor.