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

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 His(1), Asp(3), Gly(4), Phe(6), Thr(7), Ser(8), Leu(10), Asp(15), Leu(19), and Leu(23). The residues most critical for biological activity included His(1), Gly(4), Thr(7), Ser(8), GIU(9), Leu(10), Leu(19), Leu(22), and Leu(23), with Asp(3), Phe(6), Seri(11), Leu(13), Asp(15), Leu(26), and Val(27) also contributing. While the importance of residues in positions analogous to His(1), Asp(3), Phe(6), Thr(7), and Leu(23) is conserved for several closely related members of this family, Leu(19) 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 Leu(19) 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.