Solution-state conformational studies of endothelin analogs

Abstract

Endothelin-1 (ET-1), a 21-residue peptide hormone containing two disulfide bridges, is the most potent endogenous mammalian vasoconstrictor known. Endothelin binds, with high specificity, to a G-protein coupled transmembrane receptor, designated ETA. Although a consensus NMR structure of the bicyclic core has been reached, the conformation of the biologically important C-terminus remains a point of controversy. An ET-1 analog, [Pen3,15-Nle 7]-ET-1, henceforth Pen-1, is a potent ETA receptor agonist. An N-methyl scan of the C-terminal residues yields a potent ETA receptor antagonist. This analog, known as Pen-2, retains the previous substitutions of Pen-1 but includes an Ile 20 → (Nme)Ile20 substitution.NMR structure ensembles for the Pen-1 analog indicate that the bicyclic core (residues 1--15) is more rigid than that of ET-1, and thus is considered to be a better model of the bioactive state. Increased rigidity of the bicyclic core also stabilizes and lengthens the helical region (from 9--16 in ET-1 to 9--18 in Pen-1). Abnormal methyl group chemical shifts observed for the ET-1 analogs are attributed to hydrophobic clustering between the sidechains of Ile19 and Trp21. N-methylation of Ile20 appears to create a backbone conformation that enhances this hydrophobic interaction, restricts the positional space of Trp 21, and prevents ETA signaling.A number of C-terminal ET-1 and Pen-2 analogs were designed and synthesized to (1) determine if the Cys residues are essential for the helix formation, (2) examine the role of the Trp21 sidechain on the shift of the upfield 19gamma2 peak, and (3) probe the N-methyl effect on the backbone conformation in the absence of other secondary structure. NMR studies indicate the presence of a stable 7-residue helical segment in a linear Pen-2 C-terminal fragment. This segment represents one of the shortest stable helices (2 turns) observed in a peptide. CD studies of the N-methylated fragments reveals an unusually large negative band at 225 → 230nm which is absent in the non-N-methylated analogs. The resulting CD difference spectra indicate the presence of a unique turn-like signature that is thermally stable in the absence of other secondary structure.