Molecular-dynamics simulations of [Met5]- and [D-Ala2,Met5]-enkephalins. Biological implication of monomeric folded and dimeric unfolded conformations.

To investigate the biologically active conformation of enkephalin, molecular-dynamics simulations were applied to [Met5]- and [D-Ala2,Met5]-enkephalins. The dynamic trajectory of monomeric extended [Met5]-enkephalin was analysed in terms of relative mobility between respective torsions of backbone chain. After 10 ps of the dynamics simulation, the conformational transition was converged into a stationary state among the beta-bend folded forms, where they are stabilized by several intramolecular hydrogen-bond formations. Similar conformational transition was also observed in the dynamics simulation of [D-Ala2,Met5]enkephalin, which is a more mu-receptor-specific peptide than [Met5]enkephalin. The geometrical correspondence between the monomeric enkephalin conformation in the stationary state and morphine molecule (a mu-specific rigid opiate) was surveyed by virtue of the triangular substructures generated by choosing three functional atoms in each molecule, and good resemblances were observed. On the other hand, the dynamics simulation of the antiparallel extended [Met5]enkephalin dimer showed a trajectory different from that of the monomeric one. Two intermolecular hydrogen bonds at Tyr1 (NH3+)...Met5(CO2-) end residues were held throughout the 100 ps simulation, the dimeric structure being consequently kept. The conformational transition of the backbone chains from the antiparallel extended form to the twisted one took place via an intermediate state. Many conformations revealed during the dynamics simulation showed that the relative orientations of each two Tyr1, Gly3, Phe4 and Met5 residues in the dimer are nearly related by a pseudo-C2-symmetry respectively, and both halves of the dimer structure could be further fitted to the monomeric folded enkephalin conformation. The monomeric and dimeric conformations of enkephalin at their stationary states are discussed in relation to the substrate-specificity for mu- and delta-opioid receptors.     

X-ray diffraction studies of enkephalins. Crystal structure of [(4''-bromo) Phe4,Leu5]enkephalin.

In order to investigate the structure-activity relationship of [Leu5]- and [Met5]enkephalins, [(4'-bromo)Phe4, Leu5]-, [(4'-bromo)Phe4, Met5]- and [Met5] enkephalins were synthesized and crystallized. The crystal structure of [(4'-bromo) Phe4, Leu5]- enkephalin was determined by X-ray diffraction method using the heavy atom method and refined to R = 0.092 by the least-squares method. The molecule in this crystal took essentially the same type I' beta-turn conformation found in [Leu5]enkephalin [Smith & Griffin (1978) Science 199, 1214-1216). On the other hand, the preliminary three-dimensional Patterson analyses showed that the most probable conformations of [(4'-bromo)Phe4,Met5]- and [Met5]enkephalins are both the dimeric extended forms. Based on these insights, the biologically active conformation of enkephalin was discussed in relation to the mu- and delta-receptors.     

Radioiodinated [D-Ala2, Met5] enkephalin. Preparation, purification by high performance liquid chromatography and binding characteristics

125I[D-Ala2, Met5] enkephalin with high specific activity (122-185 Ci/mmol) was prepared and purified by Sep-Pak C18 reverse phase cartridge followed by high performance liquid chromatography (HPLC). HPLC at pH 3.0 resolved 125I[D-Ala2, Met5] enkephalin into two fractions, which ran as a single spot in thin-layer chromatography with the same Rf values. Alkaline hydrolysates of the HPLC-purified fractions showed a single spot corresponding to monoiodotyrosine standard when analysed by thin-layer chromatography. Binding kinetics of the tracer was found to approach equilibrium after 30 min at 24 degrees. Scatchard analysis of the saturation equilibrium binding studies gave an equilibrium dissociation constant of 3.58 nM and the number of binding site of 30 fmol/mg protein. Enkephalin analogs were capable of displacing 125I[D-Ala2, Met5] enkephalin binding from the rat brain plasma membrane. The effective concentration of [D-Ala2, Met5] enkephalin and [D-Ala2, Leu5] enkephalin for 50% inhibition of 125I[D-Ala2, Met5] enkephalin binding was estimated to be 79 nM and 23 nM, respectively. Both substance P and gastrin tetrapeptide failed to displace the 125I[D-Ala2, Met5] enkephalin binding to any significant extent. The 125I[D-Ala2, Met5] enkephalin prepared by the present procedure is therefore a useful tracer. This method of preparing radioiodinated peptide may be applicable to other enkephalin analogs or neuropeptides in general.     

Importance of folded monomer and extended antiparallel dimer structures as enkephalin active conformation. Molecular dynamics simulations of [Met5]enkephalin in water

Simulations of the molecular dynamics of the [Met5]enkephalin monomer and dimer structures in water have been carried out. The dynamic trajectories have been analyzed in terms of the distances between intra- or intermolecular polar atoms. The time-correlated conformational transitions of an extended monomer structure have been converged into a stationary state among the beta-bend folded forms. However, the dynamics simulation of an extended antiparallel dimer structure has shown no noticeable conformation change. These results imply that both the beta-bend monomer and the extended dimer structures exist together as the fundamental conformation of enkephalins.     

Further characterization of the in vitro hydrolysis of [Leu]- and [Met]enkephalin in rat plasma: HPLC-ECD measurement of substrate and metabolite concentrations.

Hydrolysis of [Leu]- and [Met]enkephalin was determined in whole rat plasma in vitro by using HPLC-ECD to measure Tyr, Tyr-Gly and Tyr-Gly-Gly formation. Although [Leu]- and [Met]enkephalin did not differ in Tyr or Tyr-Gly accumulation, the amount of Tyr-Gly-Gly resulting from [Met]enkephalin hydrolysis was greater than that resulting from [Leu]enkephalin hydrolysis, and [Met]enkephalin's half-life in plasma was slightly shorter than that of [Leu]enkephalin. By comparing metabolite formation in the presence and absence of peptidase inhibitors with high selectivity for their respective enzymes, these studies demonstrated that aminopeptidase M and angiotensin converting enzyme are the major peptidases that hydrolyze enkephalins in rat plasma.     

The probability distribution of side-chain conformations in [Leu] and [Met]enkephalin determines the potency and selectivity to mu and delta opiate receptors

The structure of enkephalin, a small neuropeptide with five amino acids, has been simulated on computers using molecular dynamics. Such simulations exhibit a few stable conformations, which also have been identified experimentally. The simulations provide the possibility to perform cluster analysis in the space defined by potentially pharmacophoric measures such as dihedral angles, side-chain orientation, etc. By analyzing the statistics of the resulting clusters, the probability distribution of the side-chain conformations may be determined. These probabilities allow us to predict the selectivity of [Leu]enkephalin and [Met]enkephalin to the known mu- and delta-type opiate receptors to which they bind as agonists. Other plausible consequences of these probability distributions are discussed in relation to the way in which they may influence the dynamics of the synapse.     

Antinociceptive effect of [Met5]enkephalin semicarbazide is not affected by dipeptidyl carboxypeptidase‐I

Dipeptidyl carboxypeptidase‐I is an enzyme involved in the biological degradation of enkephalins. It has been suggested that C‐terminal amidation of enkephalins enhances their resistance to dipeptidyl carboxypeptidase‐I‐mediated biodegradation. In this study, a novel [Met5]enkephalin amide (MEA) analogue [Met5]enkephalin (ME)‐semicarbazide synthesized by another laboratory in our group was assessed for its antinociceptive effects compared with ME‐ethylamide, MEA and ME, using tail flick test. To protect the administered drugs from biodegradation, rats were pretreated with peptidase inhibitors including amastatin, phosphoramidon and captopril. Then captopril (dipeptidyl carboxypeptidase‐I inhibitor) was deleted from the peptidase inhibitors' combination for evaluating in vivo resistance of the synthetic drugs to dipeptidyl carboxypeptidase‐I. According to the results, ME‐semicarbazide and MEA were resistant enough to dipeptidyl carboxypeptidase‐I to exert their strong antinociception following intrathecal administration even in the absence of captopril, whereas the antinociceptive effects produced by ME‐ethylamide (10 nmol) were abolished in rats not pretreated with captopril, indicating that significant amounts of the ME‐ethylamide were degraded by dipeptidyl carboxypeptidase‐I. Replacement of the amide moiety of MEA with semicarbazide provides a new ME derivative, with high analgesic effects as well as more resistance to dipeptidyl carboxypeptidase‐I‐mediated biodegradation. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational states of Leu5- and Met5-enkephalins in solution

The molecular conformations of Leu5- and Met5-enkephalins in aqueous and DMSO solutions were investigated by FT-IR and laser Raman spectroscopic methods. The amide I, II, and III regions in the FT-IR spectra of Leu5- and Met5-enkephalins in aqueous solution were analyzed by performing Fourier self-deconvolution of the bands. Leu5-enkephalin in aqueous solution is found to exist in both type II beta-turn and beta-sheet structures, whereas Met5-enkephalin has a lesser tendency to form beta-turn structure in aqueous solution. It is likely that these different conformers of enkephalins might bind to different receptor types.     

Photoreactive enkephalin analogue: [D-Ala2, p-N3-Phe4-Met5]-enkephalin. Synthesis, purification by high performance liquid chromatography and characterization

A photoreactive [D-Ala2, p-N3-Phe4-Met5]enkephalin was synthesized by classical solution peptide synthetic methods. The hydroxysuccinimide ester was used in all the coupling steps in the presence of a weak base, triethylamine. The deprotected enkephalin analogue was purified on high performance liquid chromatography using a Waters, C18 muBondapak reverse phase column and its purity was assessed by thin-layer chromatography. The composition of the analogue was determined and confirmed by elemental analysis and amino acid analysis. Its photoreactivity was demonstrated by the time dependent ultraviolet spectral changes on exposure to light. Competition receptor binding showed that [D-Ala2, p-N3-Phe4-Met5]enkephalin was 4-fold more potent than [D-Ala2, Met5]-enkephalin in competing for binding to the enkephalin binding site. The data presented suggest that this photoreactive enkephalin analogue may be suitable for use in the in situ photoaffinity labeling of the enkephalin receptor.     

Synthesis and analysis of des-Met5-[D-Ala2]enkephalinamide analogs

The effects of N- and C-terminal oligoalanine insertions into des-Met5-[D-Ala2]enkephalin amide (I) on the biological activity and spatial structure were examined. The corresponding analogues were obtained by solid-phase synthesis using Sephadex LH-20 ac a polymeric support. Biological activity was assayed via changes in the pain threshold in the rat, body temperature, and also as affinity for opiate receptors. Active analogues were obtained upon modifying the carboxylic group in the tetrapeptide I with di- and tri-D-alanyls. The CD spectra of the C-derivatized analogyes were similar to those of the starting tetrapeptide I and [Met5]enkephalin, whereas the N-derivatized analogues showed essentially different CD spectra.     

Probing the opioid receptor complex with (+)-trans-superfit. I. Evidence that [D-Pen2,D-Pen5]enkephalin interacts with high affinity at the delta cx binding site.

A variety of data support the existence of an opioid receptor complex composed of distinct but interacting mu cx and delta cx binding sites, where "cx" indicates "in the complex." The ability of subantinociceptive doses of [Leu5]enkephalin and [Met5]enkephalin to potentiate and attenuate morphine-induced antinociception, respectively, is thought to be mediated via their binding to the delta cx binding site. [D-Pen2,D-Pen5]Enkephalin also modulates morphine-induced antinociception, but has very low affinity for the delta cx binding site in vitro. In the present study, membranes were depleted of their delta ncx binding sites by pretreatment with the site-directed acylating agent, (3S,4S)-(+)-trans-N-[1-[2-(4-isothiocyanato)phenyl)-ethyl]-3-methy l-4- piperidyl]-N-phenylpropaneamide hydrochloride, which permits selective labeling of the delta cx binding site with [3H][D-Ala2,D-Leu5]enkephalin. The major findings of this study are that with this preparation of rat brain membranes: a) there are striking differences between the delta cx and mu binding sites; and b) both [D-Pen2,D-Pen5]enkephalin and [D-Pen2,L-Pen5]enkephalin exhibit high affinity for the delta cx binding site.     

Nuclear magnetic resonance study of side-chain conformation of phenylalanine residue in [Met5]-enkephalin: solvent, pH, and temperature dependence.

[Met5]-Enkephalin and N-acetylphenylalanine methylamide containing (2S,3S)-[2,3-2H2]Phe were synthesized 270 MHz 1H NMR spectra of the normal and selectively deuterated species were analysed. The lower-field and higher-field beta-proton signals of the Phe4 residue of [Met5]-enkephalin were unambiguously assigned to the pro-S and pro-R protons, respectively. The same assignments apply to N-acetylphenylalanine methylamide in polar organic solvents and in 2H2O, but the alternative assignments apply in C2HCl3. For [Met5]-enkephalin, the vicinal spin coupling constants 3JalphabetaS and 3 JalphabetaR and the rotamer populations around the Calpha-Cbeta bond were determined in a variety of solvents. From the pH and temperature dependences of rotamer populations of [Met5]-enkephalin, the side-chain conformation of the Phe residue in 2H2O solution was found to be considerably different from that in (C2H3)2SO solution. Rotamer populations of the Phe4 residue of [Met5]-enkephalin in organic solvents depend on solvent polarity. As compared with the reference model molecule of N-acetylphenylalanine methylamide, the rotamer populations of Phe4 of [Met5]-enkephalin are affected possibly by steric repulsion with other residues; the rotamer I is primarily favored but the rotamer II is appreciably destabilized in weakly polar solvents.     

Synthesis and receptor binding characteristics of [D-Ala2, cysteamine 5] enkephalin, a thiol-containing probe for structural elements of opiate receptors

For the elucidation of structural elements in the opiate receptors, a thiol-containing enkephalin analog [D-Ala2, cysteamine 5]enkephalin, and its dimeric analog were synthesized and evaluated in the radio-ligand receptor binding assays using rat brain membranes. The dimeric analog was very potent in both delta and mu assays. Comparison of receptor affinities of the thiol-containing enkephalin with those of standard mu or delta receptor specific ligands suggested that the mu receptor contains an essential thiol group which may interact with the thiol group at the C-terminus of the enkephalin analog. It also appears that no metal-ion site, postulated for the delta receptors, is present in the delta binding site.     

Ligand-Mediated Dimerization of the Met Receptor Tyrosine Kinase by the Bacterial Invasion Protein InlB

The Listeria monocytogenes surface protein InlB mediates bacterial invasion into host cells by activating the human receptor tyrosine kinase Met. So far, it is unknown how InlB or the physiological Met ligand hepatocyte growth factor/scatter factor causes Met dimerization, which is considered a prerequisite for receptor activation. We determined two new structures of InlB, revealing a recurring, antiparallel, dimeric arrangement, in which the two protomers interact through the convex face of the leucine-rich repeat domain. The same contact is found in one structure of the InlB-Met complex. Mutations disrupting the interprotomeric contact of InlB reduced its ability to activate Met and downstream signaling. Conversely, stabilization of this crystal contact by two intermolecular disulfide bonds generates a constitutively dimeric InlB variant with exceptionally high signaling activity, which can stimulate cell motility and cell division. These data demonstrate that the signaling-competent InlB-Met complex assembles with 2:2 stoichiometry around a back-to-back InlB dimer, enabling the direct contact between the stalk region of two Met molecules.     

Reserpine-induced alterations in the processing of proenkephalin in cultured chromaffin cells. Increased amidation

We have used antisera directed towards eight different portions of the proenkephalin molecule to examine the processing rates and patterns of proenkephalin-derived peptides in chromaffin cell cultures in the presence and absence of reserpine. Reserpine treatment produced profound effects on the molecular weight profile of nearly all enkephalin-containing peptides. Increased production of low molecular weight immunoreactive [Met5]enkephalin, [Leu5]enkephalin, [Met5]enkephalin-Arg6-Gly7-Leu8, and [Met5]enkephalin-Arg6-Phe7 was observed in reserpine-treated cultures; immunoreactivity corresponding to several intermediate sized enkephalin-containing peptides such as Peptide B and the high molecular weight [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactive peptide was decreased. The production of two amidated opioid peptides, amidorphin and metorphamide, was greatly accelerated in the presence of reserpine. The increased levels of low molecular weight enkephalins could not be accounted for by assuming decreased basal release. These results indicate that reserpine treatment is able to increase the extent of post-translational processing of proenkephalin within chromaffin cells.     

Evidence for a folded conformation of methionine- and leucine-enkephalin in a membrane environment

Transfer of an aqueous-soluble peptide hormone or neurotransmitter such as [Met]- or [Leu]enkephalin (Tyr1-Gly2-Gly3-Phe4-Met5(Leu5)), to the lipid-rich environment of its membrane-embedded receptor protein may convert the peptide into a ("bioactive") conformation required for eliciting biological activity. We have examined by high-resolution nuclear magnetic resonance (NMR) spectroscopy the conformational parameters of free enkephalin in aqueous solution versus those of enkephalin bound to lysophosphatidylcholine micelles using two approaches: 1) exchange rates, line broadening, coupling constants, and chemical shift changes of enkephalin backbone peptide N-H protons were measured for free and membrane-bound peptide in H2O (360 MHz, pH 5.6, 20 degrees C). A selective upfield shift observed for the Met5(Leu5) N-H proton upon lipid binding was interpreted in terms of its incorporation into an intramolecular H-bond. 2) 13C chemical shift changes induced by the shift reagent praseodymium nitrate (Pr(NO3)3) were compared in the presence and absence of lipid micelles. Significant changes occurring in Gly2 carbon atoms in membrane-bound enkephalin suggested the relative proximity of this residue to the Pr3+ atom (bound to the Met5(Leu5) COOH-terminal carboxylate 4 residues away). These combined results, in conjunction with studies on the specific interactions of enkephalin substituents with the micelles (Deber, C. M., and Behnam, B. A., (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 61-65) suggest that enkephalin folds into an intramolecularly H-bonded beta-turn structure (with an H-bond between Gly2 C = O and Met5 NH) in the lipid environment. Such folding could facilitate the positioning of strategic residues in vivo as the hormone diffuses toward its receptor.     

Comparative structure-function studies with analogs of dynorphin-(1-13) and [Leu5]enkephalin

Analogs of dynorphin-(1-13) with modifications in the enkephalin segment were compared with correspondingly modified analogs of [Leu5]enkephalin in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assay as well as in mu- and delta-receptor selective binding assays. The obtained results indicate that a) the enkephalin binding domain of the dynorphin (kappa) receptor has structural requirements which are distinct from those of the enkephalin binding site at the mu-receptor and b) the introduction of an identical conformational constraint in [Leu5]enkephalin and in the enkephalin segment of dynorphin-(1-13) produces a superpotent agonist in both cases. Fluorescence energy transfer measurements with the active [4-tryptophan]analogs of dynorphin-(1-13) and [Leu5]enkephalin and with dynorphin-(1-17) demonstrated a more extended conformation of the N-terminal tetrapeptide segment in [Trp4]dynorphin-(1-13) than in [Trp4, Leu5]enkephalin as well as the absence of an interaction between the N- and C-terminal segments of dynorphin-(1-17).     

Dimeric pentapeptide enkephalin: a novel probe of delta opiate receptors

A dimeric pentapeptide enkephalin (DPE2) consisting of two molecules of [D-Ala 2, Leu 5] enkephalin linked at C-terminal leucine with ethylenediamine, (H-Tyr-D-Ala-Gly-Phe-Leu-NH-Ch2)2 is a bivalent ligand for the delta enkephalin receptors of rat brain and neuroblastoma-glioma hybrid (NG108-15) cells. This new enkephalin analog shows dramatically increased affinity in radioligand assays using whole brain membranes when delta but not mu specific radioligands are employed. When membranes from NG108-15 cells are used, the dimer shows greatly increased activity irrespective of the mu or delta specificity of the tracer. The dimer DPE2 shows a four-fold, "sodium shift" in its IC50 for competition with [3H]naloxone, suggestive of agonist behavior. Agonist activity was confirmed by demonstrating that DPE2 inhibits cyclic AMP production in prostaglandin E1 stimulated NG108-15 cells, and by demonstrating very high potency in the mouse vas deferens bioassay. DPE2 binds to the same delta sites as the delta-selective monomer [D-Ala2, D-Leu5] enkephalin, since the two ligands show complete crossdisplacement. Radiolabeled 3H-DPE2 shows a five-fold higher affinity constant, a 2.5-fold higher association rate constant, and a two-fold lower dissociation rate than the monomer. These results are consistent with the hypothesis that the dimeric pentapeptide enkephalin can bridge two delta receptors. This enkephalin dimer provides a valuable new probe of opiate receptors and their organization in cell membranes.     

Enkephalin is liberated from metorphamide and dynorphin A1-8 by endo-oligopeptidase A, but not by metalloendopeptidase EC 3.4.24.15.

It has been previously reported that both the cysteinyl-endo-oligopeptidase A and the metalloendopeptidase EC 3.4.24.15 are able to generate enkephalin from a number of enkephalin-containing peptides, including dynorphin A1-8. The present study shows that only endo-oligopeptidase A is able to generate [Leu5]enkephalin and [Met5]enkephalin from dynorphin A1-8 and from metorphamide respectively. It is also shown that endo-oligopeptidase A neither hydrolyses the specific EC 3.4.24.15 substrate alpha-N-benzoyl-Gly-Ala-Ala-Phe p-aminobenzoate, nor is inhibited by the specific EC 3.4.24.15 inhibitor N-[1(RS)-carboxy-2-phenylethyl]-alpha-Ala-Ala-Phe p-aminobenzoate.     

Identification of the octapeptide [Met]enkephalin -Arg6-Gly7-Leu8 in extracts of bovine adrenal medulla

The primary structure of the 5300 dalton adrenal enkephalin-containing polypeptide was shown to contain at its carboxyl terminus the sequence -Lys-Arg-Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu-COOH (Jones $\text{et al}$., (1981) Proc. Natl. Acad. Sci. USA, in press). From knowledge of the type of processing that occurs at paired basic amino acid residues such as -Lys-Arg-, it was predicted that the octapeptide Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu should be produced and exist in free form in the adrenal gland. This octapeptide has now been purified from bovine adrenal chromaffin granules. Its structure was determined by amino acid analysis, carboxypeptidase Y time course hydrolysis and sequential digestion with trypsin and carboxypeptidase B. The octapeptide has 35% the opiate receptor binding activity of [Met]enkephalin.