Structure-function analysis of a series of glucagon-like peptide-1 analogs

We have used NMR in conjunction with measurements of functional bioactivity to define the receptor-binding structure of glucagon-like peptide-1 (GLP-1 .) Identification of the important residues for binding was accomplished by the substitution of amino acids at sites that seemed likely from an examin ation of the amino acid sequence and from previously published observations , to be important in the three-dimensional (3D) structure of the molecule. Identification of the receptor-bound conformation of GLP-1, because it is a flexible peptide, required constraint of the peptide backbone into a prede termined 3D structure. Constraint was achieved by the introduction of disul fide bonds and specific side chain-side chain cross-links. The biological r elevance of the synthetic structure of each rigidified peptide was assessed by measurement of its ability to bind to the receptor present on RINm5F ce lls and to elicit a functional response, cyclic AMP production. NMR solutio n structures were obtained for the most biologically relevant of these anal ogs. The results of this study indicated that the residues necessary for th e biological activity of GLP-1 occupy approximately three equally-spaced re gions of the peptide 3D structure, at the corners of an equilateral triangl e whose sides are, at a minimum estimate, 12-15 Angstrom.