Lactam Constraints Provide Insights into the Receptor-Bound Conformation of Secretin and Stabilize a Receptor Antagonist

• Published in: Biochemistry (Easton) 2011-09, Vol.50 (38), p.8181-8192
• Main Authors: Dong, Maoqing, Te, Jerez A, Xu, Xiequn, Wang, Jinhui, Pinon, Delia I, Storjohann, Laura, Bordner, Andrew J, Miller, Laurence J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites; CHO Cells; Cricetinae; Cricetulus; Humans; In Vitro Techniques; Lactams - chemistry; Ligands; Models, Molecular; Molecular Dynamics Simulation; Molecular Sequence Data; Multiprotein Complexes; Peptides - chemistry; Peptides - genetics; Peptides - metabolism; Peptides - pharmacology; Protein Conformation; Protein Stability; Receptors, G-Protein-Coupled - antagonists & inhibitors; Receptors, G-Protein-Coupled - chemistry; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Receptors, Gastrointestinal Hormone - antagonists & inhibitors; Receptors, Gastrointestinal Hormone - chemistry; Receptors, Gastrointestinal Hormone - genetics; Receptors, Gastrointestinal Hormone - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Secretin - analogs & derivatives; Secretin - chemistry; Secretin - genetics; Secretin - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi2008036

The natural ligands for family B G protein-coupled receptors are moderate-length linear peptides having diffuse pharmacophores. The amino-terminal regions of these ligands are critical for biological activity, with their amino-terminal truncation leading to production of orthosteric antagonists. The carboxyl-terminal regions of these peptides are thought to occupy a ligand-binding cleft within the disulfide-bonded amino-terminal domains of these receptors, with the peptides in amphipathic helical conformations. In this work, we have characterized the binding and activity of a series of 11 truncated and lactam-constrained secretin(5–27) analogues at the prototypic member of this family, the secretin receptor. One peptide in this series with lactam connecting residues 16 and 20 [c­[E16,K20]­[Y10]­sec­(5–27)] improved the binding affinity of its unconstrained parental peptide 22-fold while retaining the absence of endogenous biological activity and competitive antagonist characteristics. Homology modeling with molecular mechanics and molecular dynamics simulations established that this constrained peptide occupies the ligand-binding cleft in an orientation similar to that of natural full-length secretin and provided insights into why this peptide was more effective than other truncated conformationally constrained peptides in the series. This lactam bridge is believed to stabilize an extended α-helical conformation of this peptide while in solution and not to interfere with critical residue–residue approximations while docked to the receptor.