Quantitative analysis of T cell receptor complex interaction sites using genetically encoded photo-cross-linkers.
Quantitative analysis of T cell receptor complex interaction sites using genetically encoded photo-cross-linkers.
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DOI：
10.1021/cb500351s
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发表时间：
2014-09-19
影响因子：
4
通讯作者：
Krogsgaard, Michelle
中科院分区：
文献类型：
--
作者：
Wang, Wenjuan;Li, Tianqi;Felsovalyi, Klara;Chen, Chunlai;Cardozo, Timothy;Krogsgaard, Michelle
T 细胞受体 (TCR)-分化簇 3 (CD3) 信号复合物在适应性免疫反应的启动中发挥着重要作用，但较弱的相互作用阻碍了 T 细胞信号传导过程中单个 TCR-CD3 亚基相互作用的描述。在这里，我们证明非天然氨基酸 (UAA) 可用于细胞表面 TCR-CD3 亚基的光交联。将 UAA 掺入哺乳动物细胞通常是一个低效率的过程。此外，TCR-CD3由八个亚基组成，TCR和CD3链都是在细胞表面表达所必需的。由于在细胞中转染和表达多亚基蛋白复合物的困难以及 UAA 掺入效率低，用于研究 TCR-CD3 等蛋白复合物的 UAA 光交联具有挑战性。在这里，我们证明通过系统优化，我们可以将UAA高效地整合到TCR-CD3中。因此，掺入的 UAA 可用于位点特异性光交联实验，以查明蛋白质相互作用位点，并确认通过 X 射线晶体学鉴定的相互作用位点。我们系统地比较了两种不同的光交联剂——对叠氮基-苯丙氨酸 (pAzpa) 和 H-p-Bz-Phe-OH (pBpa)——它们在 2B4 TCR 中绘制蛋白质亚基相互作用的能力。 pAzpa被发现具有更高的交联效率，这表明优化最佳交联剂的选择对于正确识别蛋白质-蛋白质相互作用非常重要。因此，该方法适用于研究与各种细胞膜系统相关的大型动态异聚蛋白复合物的相互作用位点。
The T cell receptor (TCR)-cluster of differentiation 3 (CD3) signaling complex plays an important role in initiation of adaptive immune responses, but weak interactions have obstructed delineation of the individual TCR-CD3 subunit interactions during T cell signaling. Here, we demonstrate that unnatural amino acids (UAA) can be used to photo-cross-link subunits of TCR-CD3 on the cell surface. Incorporating UAA in mammalian cells is usually a low efficiency process. In addition, TCR-CD3 is composed of eight subunits and both TCR and CD3 chains are required for expression on the cell surface. Photo-cross-linking of UAAs for studying protein complexes such as TCR-CD3 is challenging due to the difficulty of transfecting and expressing multisubunit protein complexes in cells combined with the low efficiency of UAA incorporation. Here, we demonstrate that by systematic optimization, we can incorporate UAA in TCR-CD3 with high efficiency. Accordingly, the incorporated UAA can be used for site-specific photo-cross-linking experiments to pinpoint protein interaction sites, as well as to confirm interaction sites identified by X-ray crystallography. We systemically compared two different photo-cross-linkers—p-azido-phenylalanine (pAzpa) and H-p-Bz-Phe-OH (pBpa)—for their ability to map protein subunit interactions in the 2B4 TCR. pAzpa was found to have higher cross-linking efficiency, indicating that optimization of the selection of the most optimal cross-linker is important for correct identification of protein–protein interactions. This method is therefore suitable for studying interaction sites of large, dynamic heteromeric protein complexes associated with various cellular membrane systems.
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