Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R).
Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R).
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DOI：
10.1074/jbc.m117.779496
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发表时间：
2017-04-28
期刊：
影响因子：
--
通讯作者：
Sakmar TP
中科院分区：
文献类型：
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作者：
Koole C;Reynolds CA;Mobarec JC;Hick C;Sexton PM;Sakmar TP
胰高血糖素样肽-1受体（GLP-1 R）是II型糖尿病管理中的关键治疗靶点，其作用包括调节胰岛素生物合成和分泌、促进饱腹感和保持β细胞群。与大多数B类G蛋白偶联受体（GPCR）一样，GLP-1 R的生物活性与完整、全长和功能性受体·配体复合物的分子结构之间的联系知识有限。在这项研究中，我们利用遗传密码扩增位点特异性纳入光敏氨基酸对叠氮基-L-苯丙氨酸（azF）的N-末端残基的全长功能性人GLP-1 R在哺乳动物细胞。然后进行紫外光介导的azF光解以诱导靶向光交联，以确定突变受体中叠氮基与肽毒蜥外泌肽-4的接近程度。将交联数据与GLP-1 R与毒蜥外泌肽（9-39）复合物的分离N-末端胞外结构域的晶体结构直接比较，揭示了相互作用模式的相似性和明显差异。生成的分子模型，以适应光交联的限制突出了环境条件对受体肽复合物的构象的潜在影响，包括肽的折叠动力学和二聚体和更高阶的寡聚受体多聚体的形成。这些数据表明，分离的受体区域的晶体结构可能不会给出肽/受体相互作用的完整反映，并应结合额外的实验约束，以揭示肽/受体相互作用发生在动态，天然和全长受体状态。
The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety, and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biological activity of the GLP-1R with the molecular structure of an intact, full-length, and functional receptor·ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-l-phenylalanine (azF) into N-terminal residues of a full-length functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocross-linking to determine the proximity of the azido group in the mutant receptor with the peptide exendin-4. Cross-linking data were compared directly with the crystal structure of the isolated N-terminal extracellular domain of the GLP-1R in complex with exendin(9–39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a molecular model to accommodate the photocross-linking constraints highlights the potential influence of environmental conditions on the conformation of the receptor·peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide/receptor interactions and should be combined with additional experimental constraints to reveal peptide/receptor interactions occurring in the dynamic, native, and full-length receptor state.