Insights into the impact of phenolic residue incorporation at each position along secretin for receptor binding and biological activity

Abstract

Understanding of the structural importance of each position along a peptide ligand can provide important insights into the molecular basis for its receptor binding and biological activity. This has typically been evaluated using serial replacement of each natural residue with an alanine. In the current report, we have further complemented alanine scanning data with the serial replacement of each residue within secretin-27, the natural ligand for the prototypic class B G protein-coupled secretin receptor, using a photolabile phenolic residue. This not only provided the opportunity to probe spatial approximations between positions along a docked ligand with its receptor, but also provided structure-activity insights when compared with tolerance for alanine replacement of the same residues. The pattern of sensitivity to phenolic residue replacement was periodic within the carboxyl-terminal region of this peptide ligand, corresponding with alanine replacements in that region. This was supportive of the alpha-helical conformation of the peptide in that region and its docking within a groove in the receptor amino-terminal domain. In contrast, the pattern of sensitivity to phenolic residue replacement was almost continuous in the amino-terminal region of this peptide ligand, again similar to alanine replacements, however, there were key positions in which either the phenolic residue or alanine was differentially preferred. This provided insights into the receptor environment of the portion of this ligand most critical for its biological activity. As the structure of the intact receptor is elucidated, these data will provide a guide for ligand docking to the core domain and to help clarify the molecular basis of receptor activation.

Secretin Receptor as a Target in Gastrointestinal Cancer: Expression Analysis and Ligand Development

“…While the authors did not report the Ala substitutions' capacity to modulate intrinsic activity, the current study revealed Ala substitution of Asp 3 and Phe 6 to be most detrimental to the human peptide's intrinsic activity. Similarly, substitution of positions 1, 3, 4, 7, 8, 15, and 19 of rat secretin by a phenolic residue had been shown previously to impair activation of rat SCTR [81], as was the introduction of Phe 1 , Trp 3 and Trp 8 for human SCTR in the results presented here. These results are also in agreement with the recently published cryo-EM structure of a secretin-SCTR-G s complex [21] that demonstrated close interaction of secretin amino acids 1, 3, 8 and 15 with the receptor, while no proximity was identified for the other activity-related positions mentioned above.…”

Secretin receptor as a target in gastrointestinal cancer: expression analysis and ligand development

“…While the authors did not report the Ala substitutions' capacity to modulate intrinsic activity, the current study revealed Ala substitution of Asp 3 and Phe 6 to be most detrimental to the peptide's intrinsic activity. Similarly, substitution of positions 1, 3, 4, 7, 8, 15, and 19 of rat secretin by a phenolic residue had been shown previously to impair activation of rat SCTR [67], as was the introduction of Phe 1 , Trp 3 and Trp 8 for human SCTR in the results presented here. These results are also in agreement with the recently published cryo-EM structure of a secretin-SCTR-Gs complex [21] that demonstrated close interaction of secretin amino acids 1, 3, 8 and 15 with the receptor, while no proximity was identified for the other activity-related positions mentioned above.…”

Use of Cysteine Trapping to Map Spatial Approximations between Residues Contributing to the Helix N-capping Motif of Secretin and Distinct Residues within Each of the Extracellular Loops of Its Receptor

“…However, little is known about the site of docking this motif and the receptor residues that might be adjacent to it as docked in an activated ligand-receptor complex. Replacement of the residues that contribute to this motif with a photolabile residue for photoaffinity labeling has been attempted (24,25), but there has been concern that the requirement for a relatively large and bulky hydrophobic moiety that is necessary to achieve photoactivation might have provided misleading insights into natural spatial approximations that are critical for agonist activity. In the current work, therefore, we replaced these residues with smaller cysteine residues that could be used to establish relatively short and geometrically defined disulfide bonds with cysteine residues in the receptor having the proper spatial relationship.…”