TY - JOUR
T1 - Elucidation of the active conformation of the amino terminus of receptor-bound secretin using intramolecular disulfide bond constraints
AU - Dong, Maoqing
AU - Pinon, Delia I.
AU - Bordner, Andrew J.
AU - Miller, Laurence J.
N1 - Funding Information:
This work was supported by Grants from the National Institutes of Health ( DK46577 ) and from the Fiterman Foundation . The authors thank Ms. Mary Lou Augustine for her technical assistance.
PY - 2010/10
Y1 - 2010/10
N2 - Family B G protein-coupled receptors include several potentially important drug targets, yet our understanding of the molecular basis of ligand binding to and activation of these receptors is incomplete. While NMR and crystal structures exist for peptide ligand-associated amino-terminal domains of several family members, these only provide insights into the conformation of the carboxyl-terminal region of the peptides. The amino-terminal region of these peptides, critical for biological activity, is believed to interact with the helical bundle domain, and is, therefore, unconstrained in these structures. The aim of the current study was to provide insights into the conformation of the amino terminus of secretin as bound to its receptor. We prepared a series of conformationally constrained secretin peptides containing intramolecular disulfide bonds that were predicted by molecular modeling to approximate the conformation of the analogous region of PACAP bound to its receptor that had been determined using transfer-NOE NMR techniques. Secretin peptides with pairs of cysteine residues in positions 2-7, 3-5, 3-6, 4-7, 7-9, and 4-10 were studied as linear and disulfide-bonded forms. The analog with a disulfide bond connecting positions 7-9 had binding affinity and biological activity similar to natural secretin, supporting the relevance of this constraint to its active conformation. While this feature is shared between secretin and PACAP, absence of activity in other constrained peptides in this series also suggest that there are differences between these receptor-bound conformations. It will be critical to extend similar studies to other family members to learn what structural elements might be most conserved in this family.
AB - Family B G protein-coupled receptors include several potentially important drug targets, yet our understanding of the molecular basis of ligand binding to and activation of these receptors is incomplete. While NMR and crystal structures exist for peptide ligand-associated amino-terminal domains of several family members, these only provide insights into the conformation of the carboxyl-terminal region of the peptides. The amino-terminal region of these peptides, critical for biological activity, is believed to interact with the helical bundle domain, and is, therefore, unconstrained in these structures. The aim of the current study was to provide insights into the conformation of the amino terminus of secretin as bound to its receptor. We prepared a series of conformationally constrained secretin peptides containing intramolecular disulfide bonds that were predicted by molecular modeling to approximate the conformation of the analogous region of PACAP bound to its receptor that had been determined using transfer-NOE NMR techniques. Secretin peptides with pairs of cysteine residues in positions 2-7, 3-5, 3-6, 4-7, 7-9, and 4-10 were studied as linear and disulfide-bonded forms. The analog with a disulfide bond connecting positions 7-9 had binding affinity and biological activity similar to natural secretin, supporting the relevance of this constraint to its active conformation. While this feature is shared between secretin and PACAP, absence of activity in other constrained peptides in this series also suggest that there are differences between these receptor-bound conformations. It will be critical to extend similar studies to other family members to learn what structural elements might be most conserved in this family.
KW - Active peptide conformation
KW - Family B G protein-coupled receptor
KW - Ligand binding
KW - Secretin
KW - Secretin receptor
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U2 - 10.1016/j.bmcl.2010.08.062
DO - 10.1016/j.bmcl.2010.08.062
M3 - Article
C2 - 20813522
AN - SCOPUS:77956898278
SN - 0960-894X
VL - 20
SP - 6040
EP - 6044
JO - Bioorganic and Medicinal Chemistry Letters
JF - Bioorganic and Medicinal Chemistry Letters
IS - 20
ER -