Simms John, Uddin Romez, Sakmar Thomas P, Gingell Joseph J, Garelja Michael L, Hay Debbie L, Brimble Margaret A, Harris Paul W, Reynolds Christopher A, Poyner David R
Aston University , Birmingham B4 7ET , U.K.
Coventry University , Priory Street , Coventry CV1 5FB , U.K.
Biochemistry. 2018 Aug 14;57(32):4915-4922. doi: 10.1021/acs.biochem.8b00502. Epub 2018 Jul 25.
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 Thr of the peptide making contacts with L291 and H295. I284 is likely to contact Leu and Ala of CGRP, and Leu of CGRP is at the ECL/extracellular domain boundary of CLR. The reduced potency, E, and affinity of [LeuAla]-human α CGRP are consistent with this model. Contacts between Thr of CGRP and H295 may be particularly important for receptor activation.
降钙素基因相关肽（CGRP）与降钙素受体样受体（CLR）和受体活性修饰蛋白1（RAMP1）的复合物结合。CGRP如何与这个B族受体的跨膜结构域（包括细胞外环）相互作用仍不清楚。在本研究中，使用遗传密码扩展和非天然氨基酸诱变，将光亲和交联剂对叠氮基-L-苯丙氨酸（azF）主要掺入CLR的第二个细胞外环（ECL2）中。该方法经过优化，以确保受体跨膜束附近的azF残基有效光解。在第15位用荧光素修饰的CGRP类似物用于检测紫外线诱导的交联。通过确认CGRP与CLR细胞外结构域的已知接触来验证该方法。在ECL2内，主要接触点是环上的I284和第五个跨膜螺旋（TM5）顶部的L291。在S286和L290之间的ECL2边缘以及TM3中的M223和TM6中的F349处也发现了次要接触点。结合的受体复合物的全长分子模型表明，CGRP位于TM束的顶部，肽的苏氨酸与L291和H295接触。I284可能与CGRP的亮氨酸和丙氨酸接触，CGRP的亮氨酸位于CLR的ECL/细胞外结构域边界。[亮氨酸-丙氨酸]-人αCGRP效力、E值和亲和力的降低与该模型一致。CGRP的苏氨酸与H295之间的接触可能对受体激活特别重要。
