The delta-selective opioid peptide dermenkephalin and the mu-selective hybrid peptide dermenkephalin-[1-4]-dermophin-[5-7] display strikingly different conformations despite identical tetrapeptide N-termini. A quantitative 2-D NMR and molecular modeling analysis.

The selective recognition of the aminoterminal binding pharmacophore Tyr-D-Xaa-Phe of the opioid heptapeptide dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (DRM)1, and of dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (DREK), by the mu-opioid receptor and delta-opioid receptor, respectively, depends upon the constitution / conformation of the C-terminal tripeptide. The hybrid peptide DREK-[1-4]-DRM-[5-7] is very potent at, and exquisitely selective for the mu-opioid receptor, and differs only from dermenkephalin by its C-terminal tripeptide. Comparison of the structural features of DREK-[1-4]-DRM-[5-7] and dermenkephalin by nmr analysis and molecular modeling revealed striking differences, as well in the trans (Tyr5 - Pro6) isomer (population 75%) than in the cis isomer.. Whereas the folded C-terminal tail of dermenkephalin influenced the tertiary structure of the N-terminal tetrapeptide and placed the Tyr1 and Phe3 aromatic rings in definite orientations that are best suited for the delta-receptor, there were only weak contacts, as shown by NOE data, between the aminoterminal and carboxyterminal parts of the hybrid peptide. This promoted increased flexibility of the whole backbone and relaxed orientations for the side-chains of Tyr1 and Phe3 that are compatible with the mu-receptor but unsuitable for the delta-receptor. The steric hindrance introduced by Pro6 in DREK-[1-4]-DRM-[5-7], plus the absence of large hydrophobic side-chains in positions 5 and 6 may prevent close contacts between the N-terminal and C-terminal domains and reorientation of the main pharmacophoric elements Tyr1 and Phe3.

Conformational study based on simulated annealing and 1H NMR of peptides comprising the consensus sequence Arg5-Asp-Val-Arg-Gly9: effects of the substitution Ser 12 by Ala 12 or of 3 residues deletion at the N-terminus.

A conformational search by simulated annealing has been performed on two peptides derivated from the tetradecapeptide used to isolate the Xenopus laevis skin maturation RXVRG-endoprotease. The Ala 12 derivative, obtained by substitution in the hydrophobic C terminal fragment and the undecapeptide 4-14, obtained by deletion of an acidic rich tripeptide, were studied. No unique structure has been found for the tetradecapeptide Ala 12. This structural disorganization could explain the loss of activity of the endoprotease towards the substituted peptide. For the undecapeptide, two different models in accordance with the NMR data were found. The conformational differences between these two models are located in the consensus sequence and in each case an hairpin-like conformation is observed. These results could be related to the enhanced cleavage activity of the maturation enzyme. The obtained structures are also compared with those of the original tetradecapeptide.

NMR conformational study of a model tetradecapeptide mimicking the RXVRG consensus cleavage site of a Xenopus laevis skin endoprotease.

A model tetradecapeptide, used for the purification of the RXVRG-endoprotease from Xenopus laevis skin exudate, has been studied by two-dimensional NMR, correlation (COSY) and NOE (NOESY) spectroscopy. This peptide has the 5-9 consensus sequence (RXVRG), along with an acidic moiety (1-4) and a hydrophobic domain (10-14). Variations with temperature of NH chemical shifts in a dimethyl sulfoxide solution (low thermal coefficients at residues 6, 7 and 8) and quantified NOE values from four spectra at different mixing times clearly showed a structural organization in the consensus domain with psi-angles around [-40, -10 degrees] for residues 7 and 8, and two NOE correlations of alpha HiNHi + 2 type (5-7 and 6-8). Moreover, a privileged rotamer in the side chain is established for three residues (Val2, Asp3 and Val7) and limited possibilities are discussed for seven others. Most of the folding trends were not observed in the [Ser7] derivative, underlying the relationship between the conformations and a full consensus sequence. In the model tetradecapeptide an equilibrium between two beta-turns of type I, fragments 4-7 and 5-8, seems the most probable. Comparison between this tetradecapeptide and its 4-14 fragment, also a substrate for RXVRG-endoprotease, shows that the 1-3 moiety (DVD) influences the consensus domain structure(s) and clearly stabilizes the folded one(s). Finally, two analytical methods are developed in order to determine: (1) the trifluoroacetic acid content of the peptide samples, on the basis of 19F NMR spectroscopy; (2) the mean phi- and psi-angles of each residue, from the whole set of NH/alpha H coupling constants (3JN alpha) and NOE data at a local level.

Two-dimensional 1H NMR study of a tetradecapeptide with the consensus sequence Arg5-Asp-Val-Arg-Gly9: structural effects of the outside substitution Ser12 by Ala12.

Conformation of a tetradecapeptide with a RXVRG consensus sequence, Arg5-Asp-Val-Arg-Gly9, found in several precursors of antibacterian peptides, was investigated in dimethylsulfoxide solution by proton NMR spectroscopy. Complete resonance assignments and conformational parameters were obtained through correlated (COSY) and nuclear Overhauser (NOESY) techniques. The 3J(alpha H, beta H) coupling constants and the intramolecular NOE, NH...beta H, were used to analyse the conformers around the C alpha-C beta bond and, in four cases, to obtain stereospecific assignments. Use of restraints derived from NOE connectivities and 3J(NH, alpha H) coupling constants allows the determination of a range of phi and psi dihedral angles for all the residues in the sequence. The present NMR results provide favourable evidence for the formation of two bends in the consensus sequence of the tetradecapeptide. The first one has most of the features of a Glu4-Val7 beta-turn (low temperature coefficient of the Val7NH chemical shift, Arg5 alpha H...Val7NH and Asp6NH...Val7NH NOE correlations). The second one exhibits only the Asp6 alpha H...Arg7NH and Val7NH...Arg8NH NOE interactions. These consensus sequence organizations proposed were confirmed by molecular modeling based on low potential energy structure on the [4-9] fragment with high agreement of NOE data. Overall, the substitution of Ser12 by Ala12 shifts the conformation of the hydrophobic moiety [10-14] towards a quite random coil structure in this fragment and strongly destabilizes the folded structures of the consensus domain where only one NH (Val7) is solvent-shielded opposed to three (Asp6 to Arg8) in the [Ser12] tetradecapeptide. These conformational changes could be related to the processing enzyme activities on these model oligopeptides.

Conformational studies of an undecapeptide reproducing the consensus sequence around the cleavage site of the RXVRG endoprotease from Xenopus laevis skin.

Two synthetic fragments, corresponding to the 4-9 and 4-14 sequences of a tetradecapeptide used as a model to test the RXVRG-endoprotease activity from Xenopus laevis skin, have been studied by two-dimensional nmr spectroscopies, correlated spectroscopy, and nuclear Overhauser effect (NOE) spectroscopy. Both peptides wore the 5-9 consensus sequence found in several hormonal precursors. The nmr data for the 4-9 hexapeptide did not indicate any particular organization, either in water or in dimethylsulfoxide (DMSO), whereas, the 4-14 undecapeptide, a substrate for the RXVRG endoprotease, showed, in DMSO solution, significant trends of structural organization involving the amino acids pertaining to the consensus domain. From variations of integrated NOE peaks with temperature, the apparent interproton correlation times tau c were estimated and the maxima observed with Val7, the central residue in the consensus sequence. A defined tertiary structure in that domain was also supported by medium- and long-range NOEs between Asp6 and Arg8, Glu4 and Gly9, and by the likely involvement of Arg8 and Gly9 NHs in intramolecular hydrogen bonds. Most of these observations could be rationalized by an equilibrium between a 5-8 beta-turn and a 9 > 4 H-bonded loop. The predominance of one rotamer for the C alpha-C beta bond was established in four residues. Finally, the average phi and psi angles were derived from two models taking, or not, into account variations in the correlation times along the sequence. This allowed us to discuss the artefacts generated by using an average correlation time through the whole molecule.