The major determinant of exendin-4/glucagon-like peptide 1 differential affinity at the rat glucagon-like peptide 1 receptor N-terminal domain is a hydrogen bond from SER-32 of exendin-4.
OBJECTIVE
Exendin-4 (exenatide, Ex4) is a high-affinity peptide agonist at the glucagon-like peptide-1 receptor (GLP-1R), which has been approved as a treatment for type 2 diabetes. Part of the drug/hormone binding site was described in the crystal structures of both GLP-1 and Ex4 bound to the isolated N-terminal domain (NTD) of GLP-1R. However, these structures do not account for the large difference in affinity between GLP-1 and Ex4 at this isolated domain, or for the published role of the C-terminal extension of Ex4. Our aim was to clarify the pharmacology of GLP-1R in the context of these new structural data.


METHODS
The affinities of GLP-1, Ex4 and various analogues were measured at human and rat GLP-1R (hGLP-1R and rGLP-1R, respectively) and various receptor variants. Molecular dynamics coupled with in silico mutagenesis were used to model and interpret the data.


RESULTS
The membrane-tethered NTD of hGLP-1R displayed similar affinity for GLP-1 and Ex4 in sharp contrast to previous studies using the soluble isolated domain. The selectivity at rGLP-1R for Ex4(9-39) over Ex4(9-30) was due to Ser-32 in the ligand. While this selectivity was not observed at hGLP-1R, it was regained when Glu-68 of hGLP-1R was mutated to Asp.


CONCLUSIONS
GLP-1 and Ex4 bind to the NTD of hGLP-1R with similar affinity. A hydrogen bond between Ser32 of Ex4 and Asp-68 of rGLP-1R, which is not formed with Glu-68 of hGLP-1R, is responsible for the improved affinity of Ex4 at the rat receptor.
Mann
R J
RJ
Institute of Membrane and Systems Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
Nasr
N E
NE
Sinfield
J K
JK
Paci
E
E
Donnelly
D
D
D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D000077270:Exenatide; D000067757:Glucagon-Like Peptide-1 Receptor; D018698:Glutamic Acid; D006801:Humans; D006860:Hydrogen Bonding; D007004:Hypoglycemic Agents / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D051381:Rats; D018027:Receptors, Glucagon / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine; D013329:Structure-Activity Relationship; D014162:Transfection; D014688:Venoms / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology
C000593833:GLP1R protein, human; C000593835:Glp1r protein, rat; D000067757:Glucagon-Like Peptide-1 Receptor; D007004:Hypoglycemic Agents; D010455:Peptides; D018027:Receptors, Glucagon; D011994:Recombinant Proteins; D014688:Venoms; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine; D000077270:Exenatide