Mapping the binding site of calcitonin gene-related peptide to its receptor

J. Simms¹, P. Harris², S. H. Yang², M. Brimble², R. Uddin¹, D. L. Hay³, T. P. Sakmar⁴, D. Poyner¹. ¹Life and Health Sciences, Aston University, Birmingham, United Kingdom, ²School of Chemical Sciences and School of Biological Sciences, University of Auckland, Auckland, New Zealand, ³School of Biological Sciences, University of Auckland, Auckland, New Zealand, ⁴Laboratory of Chemical Biology & Signal Transduction, The Rockefeller University, New York.

Introduction: Calcitonin gene related peptide (CGRP) is a very abundant neuropeptide, especially in sensory nerve fibres. However the structure of the bound complex is still elusive. In order to tackle this problem, we have employed an unnatural amino acid approach to understand how the CGRP binds to its receptor. We have substituted each of the residues in extracellular loop 2 (ECL2) with the amber stop codon and genetically incorporated azido-phenylalanine (AzF) into the receptor structure. Subsequent UV exposure of the bound complex revealed whether a particular position is in contact with the ligand by forming a covalent complex.

Methods: Unnatural amino acid mutagenesis was performed in HEK293T cells as described previously, using amber mutant human calcitonin receptor-like receptor, RAMP1, suppressor tRNA, and azF amino-acyl tRNA synthetase (1). Membranes from the cells were incubated with 100nM human alpha CGRP (with position 15 replaced by fluorescein) for 60min at room temperature. Samples (1ml) were put into quartz cuvettes and either exposed to 300nm light for 1hr or wrapped in aluminium foil and also placed in the light source. Samples were solubilised with 1% dodecyl maltoside for 4 hours, centrifuged and 0.15ml aliquots measured for fluorescence. Fig 1. Specific binding of 15-Fluo CGRP to mutant receptors. *, fluorescence significantly different from WT, p<0.05, 1-way ANOVA followed by Dunnett’s test. Values are means and standard errors, n=3.

Results: I284 and L291 in ECL2 are the major ligand contacts as judged by specific binding (Fig 1).

Conclusion: Using this data have generated a molecular model of the docked agonist complex. Interestingly, the final position of the ligand is consistent with the recent low resolution cryo-electon microscope structure of the calcitonin receptor (2). DRP and JS were supported by BBSRC grant BB/M007529/1

References:

1) Koole, C., et al., (2017), J. Biol. Chem., 292: 7131-7144

2) Liang, Y.L., et al., (2017), Nature, 546:118-123