Importance of each residue within secretin for receptor binding and biological activity

Abstract

Secretin is a linear 27-residue peptide hormone that stimulates pancreatic and biliary ductular bicarbonate and water secretion by acting at its family B G protein-coupled receptor. While, like other family members, the carboxyl-terminal region of secretin is most important for high affinity binding and its amino-terminal region is most important for receptor selectivity and receptor activation, determinants for these activities are distributed throughout the entire length of this peptide. In this work, we have systematically investigated changing each residue within secretin to alanine and evaluating the impact on receptor binding and biological activity. The residues most critical for receptor binding were His1, Asp3, Gly4, Phe6, Thr7, Ser8, Leu10, Asp15, Leu19, and Leu23. The residues most critical for biological activity included His1, Gly4, Thr7, Ser8, Glu9, Leu10, Leu19, Leu22, and Leu23, with Asp3, Phe6, Ser11, Leu13, Asp15, Leu26, and Val27 also contributing. While the importance of residues in positions analogous to His1, Asp3, Phe6, Thr7, and Leu23 is conserved for several closely related members of this family, Leu19 is uniquely important for secretin. We, therefore, have further studied this residue by molecular modeling and molecular dynamics simulations. Indeed, the molecular dynamics simulations showed that mutation of Leu19 to alanine was destabilizing, with this effect greater than that observed for the analogous position in the other close family members. This could reflect reduced contact with the receptor or an increase in the solvent-accessible surface area of the hydrophobic residues in the carboxyl terminus of secretin as bound to its receptor.

FIGURE 1

Binding activities of the alanine-replacement secretin analogues. Top, binding curves of increasing concentrations of secretin and alanine-replacement secretin analogues to compete for binding of radioligand [¹²⁵I-Tyr¹⁰]rat secretin-27 to secretin receptor-bearing 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. Data are presented in three groups, i.e. amino-terminal (top left panel, positions 1 to 10), mid-region (top middle panel, positions 11 to 19) and carboxyl-terminal (top right panel, positions 21 to 27) based on the positions of incorporating alanine in secretin, with the affinities in each group being illustrated in the order of high to low. Bottom, role of each residue in secretin binding to its receptor. Shown are the K_i values of each of the alanine-replacement secretin analogues and the secretin sequence illustrating the role of each residue in binding to the secretin receptor. Open circles represent residues whose replacement by alanine resulted in less than 10-fold in binding affinity comparing to native secretin. Grey and black circles represent residues whose replacement with alanine 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 native secretin. Sec, secretin.

FIGURE 2

Biological activities of the alanine-replacement secretin analogues. Top, curves of intracellular cAMP responses in secretin receptor-bearing CHO-SecR cells stimulated by increasing concentrations of the alanine-replacement secretin analogues. Data points represent the means ± S.E.M. of three independent experiments performed in duplicate, normalized relative to the maximal response to secretin. Basal and maximal cAMP levels by secretin were 4.2 ± 1.1 and 197 ± 44 pmol/million cells, respectively. Data are presented in three groups, i.e. amino-terminal (top left panel, positions 1 to 10), mid-region (top middle panel, positions 11 to 19) and carboxyl-terminal (top right panel, positions 21 to 27) based on the positions of incorporating alanine in secretin, with the potencies in each group being illustrated in the order of high to low. Bottom, role of each secretin residue in its biological activity. Shown are the EC₅₀ values of each of the alanine-replacement analogues of secretin and the secretin sequence illustrating the role of each residue in their biological activity. Open circles represent residues whose replacement by alanine resulted in less than 10-fold in biological activity comparing to native secretin. Grey and black circles represent residues whose replacement with alanine 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 native secretin. Sec, secretin.

FIGURE 3

Relationship between binding affinity and biological activity of alanine-replacement analogues of secretin. Shown is the plot of the logarithmic transformations of K_i and EC₅₀ values for these constructs, as well as their correlation coefficient.

FIGURE 4

Calculated ΔΔG (in kcal/mol) for the alanine mutation of residue 15 to 27 of secretin compared to the experimental ΔΔG. The computational ΔΔG is given for the fixed backbone simulation while the experimental ΔΔG was obtained from ΔΔG = RT ln (K_i^Ala/K_i^Sec), where R is the gas constant and T is the temperature.

FIGURE 5

Structural snapshots of the complex of secretin with the amino-terminal domain of its receptor during the 12 ns simulation. Top, superimposed structure for every 1-ns time point in the molecular dynamics simulation for the natural secretin peptide (red, left) and the L19A variant of secretin (gold, right) bound to the receptor amino terminus (shades of black and gray). The complexes started with both peptides in the same conformation. The receptors are colored black (with natural secretin docked, left) and gray (with the L19A variant docked, right), with the regions of the peptides representing amino-terminal residues 1–14 (lighter colors) and carboxyl-terminal residues 15–27 (darker colors) identified. Bottom, a structural snapshot taken at the identical time point (10 ns) in the simulations showing the approximation of residue 19 of the peptides with Leu¹⁰ and Val¹³ of the secretin receptor.

FIGURE 6

RMSD of the GPCR complex simulation. Shown are the root-mean-square deviations of the backbone of the peptide-receptor complex for Sec-SecR (red), L19A/Sec-SecR (orange), VIP-VPAC2 (blue), V19A/VIP-VPAC2 (green), and GLP1-GLP1R (black).

FIGURE 7

Minimum distances established by the peptide residues to the receptor. Shown are the minimum distances between any atom of a specific residue number in the peptide and any atom in the receptor for the entire 12-ns simulation (top) and for the last 6 ns of the simulation (bottom). The different complexes are Sec-SecR (red), L19A/Sec-SecR (orange), VIP-VPAC2 (blue), V19A/VIP-VPAC2 (green), and GLP1/GLP1R (black). The residue numbering for both panels is based on the secretin peptide.

FIGURE 8

Comparison of the roles of each secretin residue in binding and biological activity with that of other family B GPCR ligands. Top, role in binding affinity. Bottom, role in biological activity. Open circles, little importance (K_i or EC₅₀ values are less than 10-fold of that of natural ligand). Grey circles, important (K_i or EC₅₀ values are more than 10-fold but less than 100-fold of that of natural ligand). Black circles, critical (K_i or EC₅₀ values are more than 100-fold of that of natural ligand). Sec, secretin; SecR, secretin receptor; GLP1R, GLP1 receptor.