Supplementary Materials

Structure and dynamics of the CGRP receptor in apo and peptide-bound forms

Tracy M. Josephs, Matthew J. Belousoff, Yi-Lynn Liang, Sarah J. Piper, Jianjun Cao, Daniel J. Garama, Katie Leach, Karen J. Gregory, Arthur Christopoulos, Debbie L. Hay, Radostin Danev, Denise Wootten, Patrick M. Sexton

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Figs. S1 to S10
  • Tables S1 and S2
  • Captions for Movies S1 to S4
  • Caption for Data S1
  • References
MDAR Reproducibility Checklist
Data S1
A. Deuterium uptake level of continuous labelling HDX-MS of CLR coupled with RAMP1 and in the absence of the CGRP peptide (RAMP1-CLR-Apo). NA indicates 17 different peptides between HDX-MS preparations. These data correspond to Fig. 4 and Fig. S8. B. Deuterium uptake level of continuous labelling HDX-MS of CLR coupled with RAMP1 and in the presence of the CGRP peptide (RAMP1-CLR-CGRP). NA indicates different peptides between HDX-MS preparations. These data correspond to figure Fig. 4 and Fig. S8. C. Deuterium uptake level of continuous labelling HDX-MS of RAMP1 coupled with CLR and in the absence of the CGRP peptide (RAMP1-CLR-Apo). NA indicates different peptides between HDX-MS preparations. These data correspond to Fig. 4 and Fig. S9. D. Deuterium uptake level of continuous labelling HDX-MS of RAMP1 coupled with CLR and in the presence of the CGRP peptide (RAMP1-CLR-CGRP). NA indicates different peptides between HDX-MS preparations. These data correspond to Fig. 4 and Fig. S9.

Images, Video, and Other Media

Movie S1
Morph between the consensus structures of the apo (blue ribbon), CGRP-bound (pink ribbon) and CGRP-bound, Gs-bound (green ribbon; PDB: 6E3Y) CGRPRs. The morph between states is shown in purple ribbon. The first segment illustrates the conformational differences that occur at the backbone level as the structure transitions from the apo to CGRP-bound to CGRPCGRPR- Gs complex (and back). The second segment highlights the large conformational transition at the base of TMs 5 and 6, with sidechains in TMs 5, 6, and 7 displayed in x-stick format. The third segment focuses on the core of the receptor and highlights changes associated with amino acids in TMs 1, 3 and 7. The final segment is from the top of the receptor with the ECD and peptide removed. It illustrates initial changes to ECL2 that occur with peptide binding, followed by upward movement of ECL2 and large changes in TMs 1, 6 and 7 that occur upon additional engagement with the G protein. Morphs between conformations were created in Chimera (46). See also Movie S2.
Movie S2
Morph between the consensus structures of the apo (blue), CGRP-bound (pink) and CGRP-bound, Gs-bound (green; PDB 6E3Y) CGRPRs highlighting key amino acids involved in conformational transitions of the receptor core and base upon peptide and G protein binding. Amino acid sidechains are shown in x-stick format colored by heteroatom. The transitional morph between the structures is illustrated in off-white with sidechains colored by heteroatom. The first segment illustrates changes to TMs 2, 3 and 7, plus helix 8 at the base of the receptor and to TM6 at the top of the receptor. The second segment illustrates the large changes in conformation of TM6 that occur in parallel with reordering of the base polar network involving the bottoms of TMs 2 and 3, and key residues in the core of the receptor (E348, F349, Q376, Y227, M230). The third segment is a view from the top of the receptor that also illustrates the reordering of these key residues during receptor activation. Morphs between conformations were created in Chimera (46). See also Movie S1.
Movie S3
HDX-MS and 3D variance analysis of the cryo-EM data provide strongly correlated data on the conformational dynamics of apo and CGRP-bound CGRPRs. The cryo-EM density map is shown in transparent grey surface representation. HDX-MS data (200 min) are illustrated on the backbone structure of the consensus structure for each of the receptor states, in licorice ribbon format where the thickness and color corresponds to the extent of deuterium uptake (red/thick, high exchange; blue/thin, low exchange). Each of the 3 principal components of the 3D variance analysis is illustrated for the apo and then the CGRP-bound structures. The 3D variance data are displayed using the ChimeraX (52) Volume Series command. See also Movie S4.
Movie S4
3D variance analysis (3DVA) of the cryo-EM data from the apo and CGRP-bound CGRPRs. Each of the 3 principal components is shown side-by-side for the apo CGRPR and then the CGRP-bound CGRPR. The last segment of the movie displays a cut through of the density and highlights the conformational change associated with binding and unbinding of the peptide Nterminus. The 3D variance data are displayed using the ChimeraX (52) Volume Series command.