Conformational Analysis of the Thrombin Receptor Agonist Peptides SFLLR and SFLLR-NH2 by NMR: Evidence for a Cyclic Bioactive Conformation

The conformational properties of the pentapeptide Ser-Phe-Leu-Leu-Arg (P5), a human thrombin receptor-derived sequence forming part of a tethered ligand which activates the thrombin receptor, and its more active amide derivative Ser-Phe-Leu-Leu-Arg-NH₂ (P5-NH₂), have been studied by proton NMR spectroscopy in dimethylsulfoxide. Measurements of nuclear Overhauser effects, performed using two-dimensional rotating frame nuclear Overhauser (ROESY) and one-dimensional nuclear Overhauser enhancement (NOE) spectroscopy, revealed that P5 exists mainly in an extended conformation. However, proton–proton 1D-NOEs between Phe C_αH and Ser C_αH, Leu³ C_αH and Leu³ NH, and Leu⁴ C_αH and Leu⁴ NH, as well as between the Ser and Arg sidechains, also implicated a minor conformer for P5 having a curved backbone and a near-cyclic structure. In contrast to P5, measurements of NOEs and ROEs for P5-NH₂ revealed a more stabilized cyclic structure which may account for its higher biological potency. Thus strong interresidue sequential NH (i)–NH (i + 1) interactions, as well as C-terminal carboxamide to N-terminal side-chain interactions, i.e., Arg CONH₂ to Phe ring and Arg CONH₂ to Ser $C_\alpha /C_{\beta \beta '} $ , observed at lower levels of the ROESY spectrum, supported a curved backbone structure for SFLLR-NH₂. Since the higher potaency P5-NH₂ analogue adopts predominantly a cyclic structure, a cyclic bioactive conformation for thrombin receptor agonist peptides is suggested.