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.

Fig. 1

Binding activities of phenolic-replacement analogues of secretin. Topbinding curves of increasing concentrations of secretin and phenolic-replacement analogues to compete for binding of secretin radioligand to CHO-SecR cells. Values illustrated represent saturable binding as percentages of maximal binding observed in the absence of the competing peptide and are expressed as the means ± S.E.M. of duplicate values from a minimum of three independent experiments. The rank order of affinities from highest to lowest is shown on the right of the graph (top to bottom). Bottomrole of each residue in secretin on binding to its receptor. Shown are the K_i values of each of the secretin analogues incorporating a phenolic residue and the secretin sequence illustrating the role of each residue in binding to the secretin receptor. Open circles represent residues whose replacement by phenolic residues resulted in less than 10-fold in binding affinity comparing to natural secretin. Grey and black circles represent residues whose replacement with phenolic residues resulted in more than 10-fold but less than 100-fold, and more than 100-fold increase in binding affinity (dashed lines), respectively, comparing to natural secretin.

Fig. 2

Biological activities of the phenolic-replacement analogues of secretin. Topcurves of intracellular cAMP responses in CHO-SecR cells stimulated by increasing concentrations of phenolic-replacement analogues. Data points represent the means ± S.E.M. of three independent experiments performed in duplicate, normalized relative to the maximal response to secretin. The rank order of potencies from highest to lowest is shown on the right of the graph (top to bottom). Basal and maximal cAMP levels by secretin were 3.7 ± 1.3 and 186 ± 40 pmol/million cells, respectively. Bottomrole of each secretin residue on its biological activity. Shown are the EC₅₀ values of each of the secretin analogues incorporating a phenolic residue and the secretin sequence illustrating the role of each residue in their biological activity. Open circles represent residues whose replacement by phenolic residues resulted in less than 10-fold in biological activity comparing to natural secretin. Grey and black circles represent residues whose replacement with a phenolic residue resulted in more than 10-fold but less than 100-fold, and more than 100-fold increase in biological activity (dashed lines), respectively, comparing to natural secretin.

Fig. 3

Relationship between binding affinities and biological activity potencies of phenolic-replacement analogues of secretin. Shown is the plot of the logarithmic transformations of K_i and EC₅₀ values for these peptides, as well as their correlation coefficient. The points in the plot are numbered to indicate the positions of the residues in secretin. Data points representing residues having disproportionate impact on biological activity or on binding affinity are circled.

Fig. 4

Comparison of the roles of each secretin residue determined by phenolic replacement and by alanine replacement. Toprole in binding affinity. Bottomrole in biological activity. Open circlesof little importance (K_i or EC₅₀ values are less than 10-fold of that of natural ligand). Grey circlesimportant (K_i or EC₅₀ values are more than 10-fold but less than 100-fold of that of natural secretin). Black circlescritical (K_i or EC₅₀ values are more than 100-fold of that of natural secretin).

Fig. 5

Comparison of the binding affinities of each of the phenolic-replacement analogues of secretin relative to those of its Ala-replacement analogues. Shown is the plot of ratio of the logarithmic transformations of K_i values of Ala-replacement secretin peptides (log K_i^Ala) and the natural secretin (log K_i^Sec) vs. the ratio of the logarithmic transformations of K_i values for secretin peptides with phenolic substitutions (log K_i^Phenolic) and the natural secretin (log K_i^Sec). The points in the plot are numbered to indicate the positions of the residues in secretin. The line of equivalence is shown, and those data points above the line reflecting the greatest preference for a phenolic residue and those below the line reflecting the greatest preference for alanine are circled.

Fig. 6

Comparison of the potencies in stimulating cAMP responses for each of the phenolic-replacement analogues of secretin relative to that of its Ala-replacement analogues. Shown is the plot of the ratio of the logarithmic transformations of EC₅₀ values of Ala-replacement secretin peptides (log EC₅₀^Ala) and natural secretin (log EC₅₀^Sec) vs. the ratio of the logarithmic transformations of EC₅₀ values for secretin peptides with phenolic substitutions (log EC₅₀^Phenolic) and natural secretin (log EC₅₀^Sec). The points in the plot are numbered to indicate the positions of the residues in secretin. The line of equivalence is shown, and those data points above the line reflecting the greatest preference for a phenolic residue and those below the line reflecting the greatest preference for alanine are circled.