The conformation of enkephalin bound to its receptor: an “elusive goal†becoming reality (original) (raw)
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Journal of the American Chemical Society, 1994
[D-Pen2, ~-Pen~]enkephalin (DPDPE), a cyclic, constrained, highly potent, and 6 opioid receptor-selective analogue of enkephalin, has been obtained from an aqueous solution in a crystalline form suitable for X-ray analysis. It crystallizes in the triclinic space group P1. The unit cell contains three conformationally distinct molecules of DPDPE which are located with approximate 3-fold symmetry about a water channel made up of approximately 24 disordered and one ordered water molecules. There are also 13 ordered water molecules which form an intricate network of hydrogen bonds which hold the peptide molecules together in the crystal. The conformation of the 14membered ring is essentially identical for all three molecules; however, the Tyr-1 residue is conformationally different in each case. Comparison of the conformations found in the crystal with those previously determined by NMR methods in conjunction with energy calculations indicates that the most favorable conformation of the 14-membered ring in aqueous solution is similar to that in the crystal. This was interpreted to be due to the cyclic constraint in DPDPE and the high degree of solvation in the crystal structure. In addition, low-energy conformations previously determined by computational methods in attempts to determine the binding conformations of DPDPE gave conformations of the 14-membered rings which were generally similar to those found in the crystal structure. These results and previous structure-activity relationships suggest that the solid-state conformations are a useful starting point for understanding the bioactive conformation important for biological activity and 6 receptor selectivity of cyclic enkephalin analogues.
Conformational sampling of bioactive conformers: a low-temperature NMR study of15N-Leu–enkephalin
Journal of Peptide Science, 1998
Conformational studies of enkephalins are hampered by their high flexibility which leads to mixtures of quasi-isoenergetic conformers in solution and makes NOEs very difficult to detect in NMR spectra. In order to improve the quality of the NMR data, Leu -enkephalin was synthesized with 15 N-labelled uniformly on all amide nitrogens and examined in a viscous solvent medium at low temperature. HMQC NOESY spectra of the labelled Leu-enkephalin in a DMSO d6 /H 2 O) mixture at 275 K do show numerous NOEs, but these are not consistent with a single conformer and are only sufficient to describe the conformational state as a mixture of several conformers. Here a different approach to the structure -activity relationships of enkephalins is presented: it is possible to analyse the NMR data in terms of limiting canonical structures (i.e. iand k-turns) and finally to select only those consistent with the requirements of l selective agonists and antagonists. This strategy results in the prediction of a family of conformers that may be useful in the design of new l selective opioid peptides.
European Journal of Biochemistry, 2005
[DMet', Pro5]Enkephalin (I) and [DMet', Pro']enkephalinamide (11), two of the more potent opioid peptides, have been studied by 'H and I3C N M R and by theoretical calculations. Their biological activity is tentatively related to their conformational behaviour. A cis S trans equilibrium around the Phe-Pro bond is brought about by the terminal prolyl residue. The cisjtrans ratios lie around 50j50 for peptide I (55 cis in dimethylsulfoxide, 35 o/, cis in water) and around 20/80 for peptide 11, the more potent compound, in both the solvents. Almost all the signals, both in 'H and I3C spectra, give separate resonances for the cis and trans rotamers, in dimethylsulfoxide as well as in water. A temperature variation allows one to determine the rotational barrier (AG;, z 81.5 kJ/mol) for the cis $ trans interconversion. The analysis of the vicinal coupling constants and theoretical calculations suggests that Tyr-DMet-Gly-Phe-Pro exists as a class of highly puckered conformations with short end-to-end distances at r = 0.4 nm and r = 0.6 nm for the trans and cis configurations respectively. The populations of the side-chain rotamers are predominantly trans/gauche (tg) for the tyrosine residue. This feature, which is quite general in enkephalins, is related to the crucial role of the tyramine moiety for opioid activity. The orientation of the Phe-4 side-chain is independent of the spatial disposition of the following prolyl residue. pH titrations provide evidence for the existence of head-to-tail interactions. Hence, in ('H6)dimethylsulfoxide, and to a lesser extent in 'HzO, the titration of the Pro-5 residue is clearly felt on the Tyr-1 moiety, whereas the deprotonation of this latter residue leads to changes in the chemical shifts of the pyrrolidine ring protons. The B protons of the Tyr-1 and Phe-4 residues are strongly magnetically nonequivalent; the opposite is true for Met-enkephalin where they show the same chemical shifts below pH 6. The titration effects suggest that no strong conformational change occurs during the amino or carboxyl group titration and indicate an important steric hindrance on the N M R time scale for the two aromatic side chains. The 13C NMR spectra of peptide I in ('Hg)dimethylsulfoxide and in a mixture of ('H6)dimethylsulfoxidej'H20 (50/50) exhibit for almost all the resonances well-separated signals, corresponding to the cis and trans conformers. The large changes in I3C chemical shifts occurring Qn the backbone and side-chain carbons during the titration steps confirm the through-space effects already deduced from the proton titration curves. Hence, several C-a (Met-2, Phe-4) and C-p (Met-2, Pro-5) atoms are affected by both the titrations of the two terminal parts of the peptide. Such effects were not observed in Met-enkephalin or Leu-enkephalin. The I3C relaxation times for the cis and trans rotamers were compared, in ('H6)dimethylsulfoxide, under strictly the same conditions. All the NTl values are small and lie in the same range both for the carbons of the peptide backbone and for the carbons of the lateral chains. The restriction in the segmental motion of the sidechains is shown by the NTl values of the 13C of the pyrrolidine ring and by the C-fl and C-y of the methionine residue which lie near those of the backbone carbons. No significant internal rotation of the aromatic ring of the tyrosyl residue seems to occur. A restricted degree of freedom for the C-a-C-p carbons is shown for the phenylalanyl residue which contrasts with Metenkephalin, Leu-enkephalin and a larger peptide such as a-endorphin.
International Journal of Peptide and Protein Research, 2009
The solution structures of a series of conformationally restricted pentapeptides with a sequence H-Tyr1-Cys2-Gly3 Phe4-Cys5-OH cyclic (2-5) disulfide, where the cysteines possess either the D or L configuration, were examined by a combined approach including NMR measurements as well as MD calculations. It turned out that at least one low energy conformer of H-Tyr1-Cys2-Gly3-Phe4-Cys5-OH cyclic (2-5) disulfide (DCDCE), as well as one conformer out of the group of calculated conformers for H-Tyr1-D-Cys2-Gly3-Phe4-Cys5-OH cyclic (2-5) disulfide (DCLCE), satisfies the NMR data obtained in this study, whereas for the derivative H-Tyrl-Cys2-Gly3-Phe4-Cys5-OH cyclic (2-5) disulfide, which contains solely L-Cys (LCLCE), there is no single structure compatible with the NMR data. © Munksgaard 1996.
International Journal of Peptide and Protein Research, 2009
Conformational analyses of the cyclic opioids H-Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE) and H-Tyr-D-Cys-Gly-Phe-D-Cys-OH (DCDCE) have been performed using the AMBER program. DPDPE is considerably more selective for delta-receptors than DCDCE. Using the RNGCFM program, a large number of ways were found to close the 14-membered disulfide-containing ring structure. However, intramolecular hydrogen bonds were only possible in gamma-turn and inverse gamma-turn conformations centered on the glycine residue which were associated with opposite chiralities of the disulfide bond. With the cyclic part of the molecules in either a gamma-turn or inverse gamma-turn, a systematic conformational analysis was performed on the tyrosine and phenylalanine sidechains. This showed that conformers with the tyrosine and phenylalanine phenyl rings in the vicinity of the disulfide bond were preferred due to attractive van der Waals forces. For DPDPE, however, this was only possible with a positive dihedral angle for the disulfide bond due to the presence of the beta-carbon methyls of Pen'. In contrast, these preferred conformers were possible with both chiralities of the disulfide bond in DCDCE. Conformational entropies and free energies were computed from the translational, rotational, and vibrational energy levels available to each conformer. The conformational entropies were found to vary significantly and to result in a reordering of the lowest energy minima. Based on these conformational differences in DPDPE and DCDCE and their differing pharmacological selectivities, tentative conformational preferences for delta-and mu-receptor opioid peptides are proposed.
Conformational preferences of a few enkephalin unsaturated analogs
Journal of Molecular Structure, 1994
The conformationalbehaviorof enkephalinanalogscontaining0'-(3 unsaturatedresidueswas studiedemploying a recentmodification (G. Alagona,C. Ghio and C. Pratesi,J. Comput.Chem.,12 (1991) 934)of an existing force field for nucleicacidsandproteins(S., J. Am. Chern.Soc., 106 (1984) 765) with molecularmechanicsand moleculardynamicssimulations. On the basis of the structuresobtained,the rationaleproposedfor the morphine-likeactivity of enkephalins(i.e. the presenceof j-i-turn of type II', consideredimportantfor the binding to opiatereceptors)was checkedandconfirmedon the basis of topological features associatedwith a compact positioning of the aromatic side chains, tyrosine and phenylalanineor dehydrophenylalanine. The molecularelectrostaticpotential in the plane perpendicularto the a -i double bond may account only in part for the enhancedpotency often observedin unsaturatedcompounds.and attributedto the intrinsic reactivity of the doublebond toward nucleophilicsites onthe opiatereceptoror to a stronger binding to receptors.In the presence of the solvent,describedas acontinuousdielectricmedium,mostof the leaststable conformationsin vacuo are greatly stabilized,thus becomingevenmore favored than the gas-phase minimum-energy structures.Interestinglyenough,the solventstabilizationis noticeablenot only for theextendedconformers,as expected. but also for several.a-turn structuresof type II'.
Conformational studies of diastereomeric cyclic enkephalins by1H-NMR and computer simulations
Biopolymers, 1987
We report the solid-phase synthesis and conformational analysis of a 14-membered, cyclic enkephalin analog, H-Tyr-~$-~-A,bu-Gly-Phe-~-Leu-] (where A,bu represents a,ydiaminobutyric acid). The results from the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays show that the analog, though active, has little seledivity for the p or 8 opioid receptors. Conformational analysis is carried out using 'H-nmr and computer simulations, including molecular dynamics and energy minimizations. The results obtained here are compared with the findings of our studies carried out on the p-receptor-selective diastereomer, Chem. SOC. 101,400&4013]. This comparison allows for insight into the regiospecificity of these cyclic enkephalin analogs. H-Tyr-~[-D-A,b~-Gly-Phe-Leu-] [N. M d , M. H m , and M. Goodman (1985) J. Am.