Mapping the Ligand-Binding Site on a G Protein-Coupled Receptor (GPCR) Using Genetically Encoded Photocrosslinkers
Mapping the Ligand-Binding Site on a G Protein-Coupled Receptor (GPCR) Using Genetically Encoded Photocrosslinkers
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DOI：
10.1021/bi200214r
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发表时间：
2011-05-03
期刊：
影响因子：
2.9
通讯作者：
Sakmar, Thomas P.
中科院分区：
文献类型：
--
作者：
Grunbeck, Amy;Huber, Thomas;Sakmar, Thomas P.
我们开发了一种通用的基于细胞的光交联方法来研究G蛋白偶联受体（GPCR）信号复合物形成所需的结合界面。通过琥珀密码子抑制技术将两种可光活化的非天然氨基酸对苯甲酰基-L-苯丙氨酸和对叠氮基-L-苯丙氨酸整合到CXC趋化因子受体4（CXCR 4）中。然后，我们使用荧光素标记的T140类似物探测HIV-1辅助受体阻断剂T140的配体结合位点。在八个氨基酸的位置测试，我们发现了一个独特的紫外光依赖性交联，特别是残基189和T140之间。使用与CVX 15结合的CXCR 4的晶体结构，通过分子建模评估这些结果。
We developed a general cell-based photocrosslinking approach to investigate the binding interfaces necessary for the formation of G protein-coupled receptor (GPCR) signaling complexes. The two photoactivatable unnatural amino acids p-benzoyl-l-phenylalanine and p-azido-l-phenylalanine were incorporated by amber codon suppression technology into CXC chemokine receptor 4 (CXCR4). We then probed the ligand-binding site for the HIV-1 coreceptor blocker, T140, using a fluorescein-labeled T140 analogue. Among eight amino acid positions tested, we found a unique UV-light-dependent crosslink specifically between residue 189 and T140. These results are evaluated with molecular modeling using the crystal structure of CXCR4 bound to CVX15.