Photoaffinity Cross-Linking and Unnatural Amino Acid Mutagenesis Reveal Insights into Calcitonin Gene-Related Peptide Binding to the Calcitonin Receptor-like Receptor/Receptor Activity-Modifying Protein 1 (CLR/RAMP1) Complex.
Photoaffinity Cross-Linking and Unnatural Amino Acid Mutagenesis Reveal Insights into Calcitonin Gene-Related Peptide Binding to the Calcitonin Receptor-like Receptor/Receptor Activity-Modifying Protein 1 (CLR/RAMP1) Complex.
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DOI：
10.1021/acs.biochem.8b00502
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发表时间：
2018-07
期刊：
影响因子：
2.9
通讯作者：
J. Simms;Romez Uddin;T. Sakmar;Joseph J Gingell;Michael L Garelja;D. Hay;M. Brimble;P. Harris;C. Reynolds;D. Poyner
中科院分区：
文献类型：
--
作者：
J. Simms;Romez Uddin;T. Sakmar;Joseph J Gingell;Michael L Garelja;D. Hay;M. Brimble;P. Harris;C. Reynolds;D. Poyner
降钙素基因相关肽（CGRP）与降钙素受体样受体（CGRP）和受体活性修饰蛋白1（RAMP 1）的复合物结合。CGRP如何与该家族B受体的跨膜结构域（包括细胞外环）相互作用仍不清楚。在这项研究中，一个光亲和交联剂，对叠氮基L-苯丙氨酸（azF），被纳入到ESTA，主要是在第二个细胞外环（ECL 2）使用遗传密码扩展和非天然氨基酸诱变。优化该方法以确保受体跨膜束附近azF残基的有效光解。在位置15处用荧光素修饰的CGRP类似物用于检测紫外线诱导的交联。该方法通过确认已知的CGRP与CGRP胞外结构域的接触来验证。在ECL 2中，主要接触点是环本身上的I284和第五跨膜螺旋（TM 5）顶部的L291。沿着S286和L290之间的ECL 2边缘观察到轻微接触，并且还与TM 3中的M223和TM 6中的F349发生接触。结合受体复合物的全长分子模型表明CGRP位于TM束的顶部，肽的Thr 6与L291和H295接触。I284可能与CGRP的Leu 12和Ala 13接触，CGRP的Leu 16位于ECL/胞外域边界。[Leu 16 Ala]-人α CGRP的降低效价、Emax和亲和力与该模型一致。CGRP的Thr 6和H295之间的接触可能对受体活化特别重要。
Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). How CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor remains unclear. In this study, a photoaffinity cross-linker, p-azido l-phenylalanine (azF), was incorporated into CLR, chiefly in the second extracellular loop (ECL2) using genetic code expansion and unnatural amino acid mutagenesis. The method was optimized to ensure efficient photolysis of azF residues near the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of ultraviolet-induced cross-linking. The methodology was verified by confirming the known contacts of CGRP to the extracellular domain of CLR. Within ECL2, the chief contacts were I284 on the loop itself and L291, at the top of the fifth transmembrane helix (TM5). Minor contacts were noted along the lip of ECL2 between S286 and L290 and also with M223 in TM3 and F349 in TM6. Full length molecular models of the bound receptor complex suggest that CGRP sits at the top of the TM bundle, with Thr6 of the peptide making contacts with L291 and H295. I284 is likely to contact Leu12 and Ala13 of CGRP, and Leu16 of CGRP is at the ECL/extracellular domain boundary of CLR. The reduced potency, Emax, and affinity of [Leu16Ala]-human α CGRP are consistent with this model. Contacts between Thr6 of CGRP and H295 may be particularly important for receptor activation.