Figure 1.
HDX of Peptide Ligands Color Coded onto Crystal Structure.
Average % deuterium exchange values for exendin-4 and exendin-4[9-39] overlaid onto the crystal structure pdb:3C5T. Deuterium exchange was measured in the presence of DDM/CHS micelles as indicated in the Experimental section. The nGLP-1R was not present in these samples, but is included in gray for visual reference. As shown in the key, color indicates the average % deuterium uptake across all five time points (10 s to 1 hr) for digested peptic peptides within each region of the peptide ligands. Deuterium exchange at additional residues specific to the agonist (not present in the antagonist crystal structure shown) are indicated with the rectangular box on the N-terminal side of the ligand.
Figure 2.
Stabilization of Peptide Ligands by nGLP-1R.
A ‘perturbation map’ showing changes to deuterium exchange for peptide ligands in the presence and absence of nGLP-1R. Deuterium exchange was measured in the presence of DDM/CHS micelles as indicated in the Experimental section. Resolved peptides are indicated with rectangular boxes below the sequence of exendin-4 and exendin-4[9-39]. The whiskers on the left side of each bar indicate amino acids that are included in the detected peptides, but whose amides do not report on deuterium in-exchange. As a result of digestion the first amino acid no longer contains an exchangeable amide and the back exchange rates are higher for the second amino acid. The average % change in deuterium in the presence of nGLP-1R is included inside the boxes with standard error in parenthesis. Boxes are colored according to the key where significant changes are determined by t-test. The peptide enclosed in the red box was chosen for ETD fragmentation.
Figure 3.
HDX at Single Amino Acid Resolution for Exendin-4[9-39].
A) Top: Crystal structure of exendin-4[9-39] (teal) bound to nGLP-1R (Gray), pdb3C5T. Bottom: The location of the backbone amide hydrogen bond on Asn28 is shown in green in the zoomed in image. B) Deuterium exchange on individual amino acids from ETD data for exendin-4[9-39] in the absence and presence of nGLP-1R. Deuterium exchange was measured in the presence of DDM/CHS micelles as indicated in the Experimental section. The average number of deuterium atoms exchanged is shown for each c fragment ion with the corresponding amino acid. Note that the c6 amide contains the Lys27 side chain, but the amide is associated with Asn28 in the sequence. A detailed structure of the peptide and c fragments is shown below the ETD data for reference. Significant increases in deuterium exchange as determined by Tukey analysis are indicated with vertical transitions on the orange lines. Each deuterium exchange measurement is the average of 5 replicates after 30 s of exchange.
Figure 4.
Changes to HDX of nGLP-1R in the Presence of Peptide and Small Molecule Ligands.
A) The WETVQKWRE peptide of nGLP-1R where protection was observed is shown in red on the crystal structure pdb:3C5T. Deuterium exchange was measured in the presence of DDM/CHS micelles as indicated in the Experimental section The exendin-4[9-39] peptide ligand is colored gray. B) Table showing the average change in % Deuterium in this same nGLP-1R peptide (WETVQKWRE) for four peptide ligands and four proposed small molecule ligands. GLP-1 induced cAMP accumulation was quantified in HEK293 cells transfected with plasmids expressing either (C) the GLP-1R or (D) a GIPR/GLP-1R chimeric protein where the GLP-1R 7TM domains are fused in-frame with the GIP-R ectodomain. Dose response curves of GLP-1 were generated and robust cAMP accumulation was observed for the GLP-1R (EC50 = 9.0 pM) and GIPR/GLP-1R (1.3 nM). The ability of 6-BPPI to antagonize GLP-1 signaling was measured by treatment of cells with a dose-response of 6-BPPI in the presence of EC50 to EC80 concentrations of GLP-1. Dose-dependent blockade of GLP-1R signalling by 6-BPPI (100 pM GLP-1, IC50 = 310 nM; 30 pM GLP-1, IC50 = 110 nM, 10 pM GLP-1, IC50 = 33 nM) but not GIPR/GLP-1R was observed.