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

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 1284 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 5286 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(6) of the peptide making contacts with L291 and H295. 1284 is likely to contact Leu(12) and Ala(13) of CGRP, and Leu(16) of CGRP is at the ECL/extracellular domain boundary of CLR. The reduced potency, E-max, and affinity of [Leu(16)Ala]-human alpha CGRP are consistent with this model. Contacts between Thr(6) of CGRP and H295 may be particularly important for receptor activation.