Molecular Approximations between Residues 21 and 23 of Secretin and Its Receptor: Development of a Model for Peptide Docking with the Amino Terminus of the Secretin Receptor

Abstract

The structurally unique amino-terminal domain of class II G protein-coupled receptors is critically important for ligand binding and receptor activation. Understanding the precise role it plays requires detailed insights into the molecular basis of its ligand interactions and the conformation of the ligand-receptor complex. In this work, we used two high-affinity, full-agonist, secretin-like photolabile probes having sites for covalent attachment in positions 21 and 23 and used sequential proteolysis and sequencing of the labeled region of the receptor to identify two new spatial approximation constraints. The position 21 probe labeled receptor residue Arg 15 , whereas the position 23 probe labeled receptor residue Arg 21 . A homology model of the amino-terminal domain of the secretin receptor was developed using the NMR structure of the analogous domain of the corticotropin-releasing factor receptor. This was attached to a homology model of the secretin receptor transmembrane bundle, with the two domains oriented relative to each other based on continuity of the peptide backbone and by imposing a distance restraint recently identified between the amino-terminal WDN sequence and the region of the helical bundle above transmembrane segment six. Secretin was docked to this model using seven sets of spatial approximation constraints identified in previous photoaffinity labeling studies. This model was found to fully accommodate all existing constraints, as well as the two new approximations identified in this work.The secretin receptor is a prototypic member of the class II family of G protein-coupled receptors (GPCRs) that contains several potentially important drug targets, such as receptors for calcitonin, vasoactive intestinal polypeptide, glucagon, glucagon-like peptide, corticotropin-releasing factor (CRF), and parathyroid hormone (Ulrich et al., 1998). Despite recent advances in the study of this receptor family, our understanding of receptor structure-function remains limited. Refinement of our understanding of the molecular basis of agonist ligand binding and activation of the secretin receptor should contribute insights relevant to the entire family and facilitate the design and refinement of potential receptoractive drugs.

Spatial Approximations between Residues 6 and 12 in the Amino-terminal Region of Glucagon-like Peptide 1 and Its Receptor

“…Another photolabile probe, [Bpa 6 ,Arg 26,34 ]GLP1(7-36) (Bpa 6 probe), was designed to incorporate a Bpa at the amino-terminal extension of the GLP1 ligand to minimize the negative functional impact on the critical residue, His 7 . Both probes contain a naturally occurring Tyr residue in position 19 as a site for radioiodination.…”

“…A signature structural feature of this family is a long and structurally complex extracellular aminoterminal domain containing six conserved cysteine residues that form disulfide bonds that contribute to the development of a highly folded structure (5)(6)(7)(8)(9). This domain has been suggested to be the predominant domain for natural ligand binding and this is a consistent theme throughout the family (10 -16).…”

Do Plants Contain G Protein-Coupled Receptors?

“…In this respect, only GCR1 is useful, as the fold recognition studies indicate that GCR1 is the most likely candidate to have a GPCR fold while the evidence for other plant GPCRs is at best minimal. While many methods have been used to align GPCRs from different classes (Frimurer and Bywater 1999;Sheikh et al, 1999;Bissantz et al, 2004;Miedlich et al, 2004;Eilers et al, 2005;Kratochwil et al, 2005;Dong et al, 2007;Coopman et al, 2011;Gregory et al, 2013), it has not been possible to validate these methods on GPCRs until recently. However, with the recent publication of the structure of the class B glucagon receptor , the class B corticotropinreleasing factor1 receptor (Hollenstein et al, 2013), and the class F human smoothened receptor (Wang et al, 2013) and the associated structural alignments between class A and these remote homologs, we have been able, to our knowledge for the first time, to successfully test our new method.…”

Molecular Basis of Glucagon-like Peptide 1 Docking to Its Intact Receptor Studied with Carboxyl-terminal Photolabile Probes

“…Interestingly, the carboxyl-terminal , and amino-terminal (position 6) probes have been demonstrated to label the distal amino terminus of the secretin receptor, all within the first 38 residues (45,46). The only secretin analogue that labels a residue (Val 103 ) within the amino-terminal domain adjacent to the transmembrane domain, in positions analogous to the GLP1 receptor residues labeled by the carboxyl-terminal position 24 and 35 GLP1 probes in this work, is the midregion position 13 probe (25).…”

“…The most extensive data for docking a natural peptide ligand in this family to its receptor come from photoaffinity labeling with analogues of secretin where spatial approximations have been established between residues scattered throughout the pharmacophore of secretin in positions 1, 5, 6, 12, 13, 14, 18, 21, 22, 23, and 26 (45,46). Interestingly, the carboxyl-terminal , and amino-terminal (position 6) probes have been demonstrated to label the distal amino terminus of the secretin receptor, all within the first 38 residues (45,46).…”