Publisher Summary Turns are a fundamental class of polypeptide structure and are defined as sites where the peptide chain reverses its overall direction. In the past 20 years, the peptide field has witnessed major development, stimulated by the discovery of a host of bioactive peptides. Turn structures have been proposed and implicated in the bioactivity of several of these naturally occurring peptides. In addition, many structural details of turns have been derived from conformational studies of model peptides. During this same period, more than 100 complete protein structures have been elucidated in single-crystal X-ray studies. These examples document the rich diversity of structural patterns in the chain folds of native proteins. Turns are intrinsically polar structures with backbone groups that pack together closely and side chains that project outward. Such an array of atoms may constitute a site for molecular recognition, and indeed, the literature abounds with suggestions that turns serve as loci for receptor binding, antibody recognition, and post-translational modification. In peptides, turns are the conformations of choice for simultaneously optimizing both backbone–chain compactness (intramolecular nonbonded contacts) and side-chain clustering (to facilitate intermolecular recognition). Presence of turns in bioactive conformations may in fact also reflect the lack of alternative conformational possibilities. The aim of this chapter is to examine structural and functional roles of turns in peptides and proteins.

Turns in peptides and proteins.

Opiate drugs have potent analgesic and addictive properties. These drugs interact with receptors that also mediate the response to endogenous opioid peptide ligands. However, the receptors for opioids have eluded definitive molecular characterization. By transient expression in COS cells and screening with an iodinated analog of the opioid peptide enkephalin, a complementary DNA clone encoding a functional delta opioid receptor has been identified. The sequence shows homology to G protein-coupled receptors, in particular the receptors for somatostatin, angiotensin, and interleukin-8.

Cloning of a delta opioid receptor by functional expression

Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled µ-opioid receptor (µ-OR) in the central nervous system. Here we describe the 2.8 A crystal structure of the mouse µ-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the µ-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.

/pdf/crystal-structure-of-the-u-opioid-receptor-bound-to-a-3114f9v1hs.pdf

Crystal structure of the µ-opioid receptor bound to a morphinan antagonist

even less so. It was therefore impossible for the functionally minded neuro­ scientist to design and execute experiments taking this heterogeneity into consideration. Even pharmacological studies with these peptides were difficult to interpret in a physiological framework. In 1982, thanks mainly to the recombinant DNA techniques, we learned that all these peptides belong to three genetically distinct peptide families, as we describe below. We now know a great deal more about their anatomy, and we are beginning to clarify their biosynthetic pathways. Suddenly, we can think in terms of circuits rather than in "humors" or in black boxes. In our study offunction, we can no longer ignore the multiple systems, any more than the student of monoamines can ignore differences between dopaminergic and noradrenergic systems. Less clear, but equally critical, is the issue of multiple opioid receptors. Unquestionably, the heterogeneity exists. What remains to be established is whether each of the three families of opioids has its own receptor, or whether a given family can interact with more than one SUbtype, and each receptor subtype with more than one family. More critical to physiology is whether

Endogenous Opioids: Biology and Function

In the guinea pig ileum myenteric plexus--longitudinal muscle preparation, dynorphin-(1--13) and the prototypical kappa agonist ethylketocyclazocine had equally poor sensitivity to naloxone antagonism and showed selective cross protection in receptor inactivation experiments with the alkylating antagonist beta-chlornaltrexamine. In binding assays with membranes from guinea pig brain, ethylketocyclazocine and dynorphin-(1--13) amide were more potent in displacing tritium-labeled ethylketocyclazocine than in displacing typical mu and delta opioid receptor ligands. In the two preparations studied, the dynorphin receptor appears to be the same as the kappa opioid receptor.

http://science.sciencemag.org/content/sci/215/4531/413.full.pdf

Dynorphin is a specific endogenous ligand of the kappa opioid receptor.

Opioid peptides were assayed by inhibition of 3H-naloxone and 3H-leu-enkephalin binding in brain homogenates and by depression of contractions of the guinea pig ileum and mouse vas deferens. We conclude that the opioid peptidergic system has agonists of different characteristics which interact with more than one type of receptor.

Endogenous opioid peptides: multiple agonists and receptors

Analogues of β-LPH61–64 posessing selective agonist activity at μ-opiate receptors

1 The activity pattern of analogues of the enkephalins was determined in four parallel assays, the inhibition of the electrically evoked contraction of the guinea-pig ileum and mouse vas deferens at 36 degrees C and the inhibition of [(3)H]-naltrexone and [(3)H]-leucine-enkephalin binding at 0 to 4 degrees C in homogenates of guinea-pig brain.2 The activity pattern was best characterized by the ratio of the potency in the guinea-pig ileum to that in the mouse vas deferens (G.p.i./M.v.d.) and the ratio of the potency in inhibiting [(3)H]-naltrexone binding to that in inhibiting [(3)H]-leucine-enkephalin binding (Nal/Leu).3 The enkephalins had low G.p.i./M.v.d. (0.02 to 0.09) and low Nal/Leu (0.05 to 0.18) ratios whereas the corresponding values for morphine were 7.0 and 7.5.4 Analogues obtained by substituting D-Ala for Gly(2) and D-Met or D-Leu for L-Met(5) or L-Leu(5) showed only minor changes in G.p.i./M.v.d. (0.01 to 0.11) and in Nal/Leu (0.06 to 0.13) ratios.5 Analogues in which resistance to enzymatic degradation was brought about by amidation of the C-terminal carboxylic group or methylation of the amino group of tyrosine or both modifications, had G.p.i./M.v.d. ratios of 1.2 to 5.5 and Nal/Leu ratios of 0.5 to 21. High values (2.1 and 3.4) were found for the potent antinociceptive analogue of Sandoz, Tyr-D-Ala-Gly-NCH(3)Phe-Met(O)-ol.6 In the mouse vas deferens, some of the analogues with high G.p.i./M.v.d. and Nal/Leu ratios were tested for antagonism by naloxone and found to require less than the high concentration needed for the natural enkephalins. C57/BL mice, which have a lowered sensitivity to morphine but a normal response to peptides with low G.p.i./M.v.d. and Nal/Leu ratios, had a lowered sensitivity to analogues with high ratios.7 In the alkaloid-like series of narcotic analgesic drugs, ketobemidone, levorphanol, methadone, etorphine and the antagonist Mr 2266 had lower Nal/Leu ratios (1.0 to 2.8) than morphine, normorphine, naloxone and naltrexone (8 to 12).8 It would appear that compounds with low G.p.i./M.v.d. and Nal/Leu ratios interact mainly with delta-receptors in the brain and peripheral nervous system while compounds with high ratios interact mainly with mu-receptors. For antinociceptive action mu-receptors may be more important than delta-receptors.

/pdf/effects-of-changes-in-the-structure-of-enkephalins-and-of-1b0w14tszk.pdf

Effects of changes in the structure of enkephalins and of narcotic analgesic drugs on their interactions with μ and δ-receptors

Opioid activities of fragments of β-endorphin and of ites leucine65-analogue. Comparison of the binding properties of methionine- and leucine-enkephalin

Appropriate modification of 14 beta-methoxy- and 14 beta-ethoxycodeinone (prepared by alkylation of 14 beta-hydroxycodeinone) has generated four alkoxy analogues (3a-d) of naloxone and naltrexone. These agents were pure narcotic antagonists in contradiction to the predictions of the common anionic receptor site hypothesis, postulated to be of importance in the enhanced antagonism of naloxone. The molecular change from allyl to cyclopropylmethyl on the N atom increased selectivity of these antagonists for the mu receptor to the same extent as found for naloxone. Increase in the length of the C14 O-substituent had no effect on receptor selectivity, and either formation in most cases did not significantly alter oral/parenteral ratios of durations of action.

John A.H. Lord

Papers

Endogenous opioid peptides: multiple agonists and receptors

Analogues of β-LPH61–64 posessing selective agonist activity at μ-opiate receptors

Effects of changes in the structure of enkephalins and of narcotic analgesic drugs on their interactions with μ and δ-receptors

Opioid activities of fragments of β-endorphin and of ites leucine65-analogue. Comparison of the binding properties of methionine- and leucine-enkephalin

Common anionic receptor site hypothesis: its relevance to the antagonist action of naloxone