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The ligand binding modes of a series of fentanyl derivatives are examined using a combination of conformational analysis and molecular docking to the mu-opioid receptor. Condensed-phase molecular dynamics simulations are applied to evaluate potential relationships between ligand conformation and fentanyl substitution and to generate probable "bioactive" structures for the ligand series. Automated docking of the largely populated solution conformers identified a common binding site orientation that places the N-phenethyl group of fentanyl deep in a crevice between transmembrane (TM) helices II and III while the N-phenylpropanamide group projected toward a pocket formed by TM-III, -VI, and -VII domains. An analysis of the binding modes indicates the most potent fentanyl derivatives adopt an extended conformation both in solution and in the bound state, suggesting binding affinity may depend on the conformational preferences of the ligands. The results are consistent with ligand binding data derived from chimeric and mutant receptor studies as well as structure-activity relationship data reported on a wide range of fentanyl analogues. The binding site model is also compared to that of N-phenethylnormorphine. An overlay of the bound conformation of the opiate and cis-3-methylfentanyl shows the N-phenethyl groups occupy equivalent binding domains in the receptor. While the cationic amines of both ligand classes were found docked to an established anchor site (D149 in TM-III), no overlap was observed between the N-phenylpropanamide group and the remaining components of the opiate scaffold. The unique binding mode(s) proposed for the fentanyl series may, in part, explain the difficulties encountered in defining models of recognition at the mu-receptor and suggest opioid receptors may display multiple binding epitopes. Furthermore, the results provide new insight to the design of experiments aimed at understanding the structural basis to the differential selectivities of ligands at the mu-, delta-, and kappa-opioid receptors.

An alignment of the transmembrane domains of halobacterial retinal proteins (including bacteriorhodopsin) and G-protein coupled receptors (GPCRs) is presented based on the commonality of conserved residues between families. Due to the limited sequence homology displayed by these proteins, an alternative strategy is proposed for sequence alignment that correlates residues within secondary structure elements. The nonsequential alignment developed identifies three proline and two aspartates residues that share common positions and, in the former case, similar functions in the transmembrane domain. The alignment is further applied to model the packing of transmembrane helices 5 and 6 of the beta-adrenergic receptor based on the backbone coordinates of bacteriorhodopsin helices 3 and 2, respectively. Unlike models derived from standard sequential alignments, the approach developed here allows the key structural features conferred by the proline residues to be captured during model building. The structure described is also compared with available site directed mutagenesis results as well as existing GPCR models. In addition to the implications to model building, the commonality observed suggests a potential relationship among the GPCRs and retinal proteins.

The delta-selective opioid antagonist naltrindole (NTI), as well as the kappa-selective opioid antagonists norbinaltorphimine (norBNI) and 5'-guanidinonaltrindole (GNTI), are derived from naltrexone, a universal opioid antagonist. Previous studies have indicated that extracellular loop III is the key region for discrimination by naltrexone-derived selective ligands between the delta, mu, and kappa opioid receptor types. It has been proposed that selective ligands could bind to all three receptor types if the appropriate portions of the extracellular loops were eliminated. To investigate this possibility, several single-point mutant opioid receptors have been generated with the aim of conferring enhanced affinity of selective ligands for their nonpreferred receptor types. Mutations were made in all three types of opioid receptors with the focus on two positions at the extracellular end of transmembrane regions (TM) VI and VII. It was found that the delta-selective NTI could bind both mu and kappa receptors with significantly enhanced affinity when an aromatic residue in TM VII was replaced with alanine (mu[W318A] and kappa[Y312A]). Similarly, kappa-selective antagonists, norBNI and GNTI, showed enhanced affinity for the mu[W318A] mutant and for both mu and delta receptors when a glutamate residue was incorporated into the extracellular end of TM VI (mu[K303E] and delta[W284E]). These results demonstrate that naltrexone-derived selective ligands achieve their selectivity via a combination of enhanced affinity of the address for a particular subsite along with loss of affinity due to steric interference at nonpreferred types. The results reveal key residues in the "address" recognition locus that contribute to the selectivity of opioid ligands and support the hypothesis that recognition of the naltrexone moiety is essentially the same for all three receptor types.

The three-dimensional structure, dynamics, and binding modes of representative kappa-opioid agonists of the arylacetamide class (U50, 488; U69,593; U62,066; CI-977; ICI199,441; ICI197,067; BRL52,537; and BRL52,656) have been investigated using molecular modeling techniques. Systematic exploration of the conformational space of the ligand combined with molecular dynamics (MD) simulations in water revealed consistent conformational preferences for all the kappa-agonists in this series. The results were further compared with available X-ray and 1D- and 2D-NMR data to identify potential "lead" conformers for molecular docking. Ligand binding modes were initially determined using automated docking of two of the ligands (U50,488 and BRL52,537) to the kappa-opioid receptor. Extrapolation of the predicted binding mode to other members in this ligand series revealed similar docking preferences, with each ligand docked along the receptor helical axis. The binding modes were further refined using MD simulations of the receptor-ligand complexes. The results show a that salt bridge is formed between the amino proton of the ligands and the carboxylate group of Asp138 in TM3. This interaction most likely serves as a key anchoring point for the agonist association. Additional ligand contacts were noted with kappa-specific residues Ile294, Leu295, and Ala298, which may, in part, explain the kappa-selectivity in this series. In comparing the arylacetamides with opiate-based ligands, no evidence was found to link these classes through a common binding motif (except for the ion pair). The binding site model was also applied to explain the enantiomeric preference of U50,488 and to provide insight to the mu/kappa-selectivity of representative ligands in this series. Overall, the results provide a structure-based rationale for ligand recognition that is consistent both with site-directed mutagenesis experiments and structure-function relationship data.

The sequence-dependent local destabilization in the interior of the collagen triple helix has been evaluated by means of conformational energy computations. Using a model poly(Gly-Pro-Pro) triple helix as the reference state, a method was developed for generating local loops, i.e., internal deformations, and analyzing their conformations. A seven-residue Gly-Pro-Pro- Gly-Pro-Pro-Gly fragment was replaced by the Gly-Pro-Ala-Gly-Ala-Ala-Gly sequence in one, two, or all three of the strands of the loop region. A set of loop conformations was generated in which the ends of the loop were initially fixed in the triple-helical structure. The potential energy of the entire deformed triple helix was then minimized, resulting in a variety of structures that contained deformed loops. The conformations of the triple helices at the two ends of the loops remained essentially unchanged in many of the low-energy conformations. In numerous high-energy conformations, however, the triple-helical segments were also partially or totally disrupted. The minimum-energy conformations of the whole structures were compared in terms of rms deviations of atomic coordinates with respect to the original triple helix, and of the shapes of the loops (using a distance function derived from differential geometry). Three new geometrical parameters-stretch S, kink K, and unwinding U-were defined to describe the changes in the overall orientation of the triple helices at the two ends of the loop. It is shown that, when the number of Pro residues in a short fragment is reduced, the triple helical structure can accomodate internal loops (i.e., distortions) within a 5 kcal/mol cutoff from the essentially unperturbed triple helical structure. For structures with a Gly-Pro-Ala-Gly-Ala-Ala-Gly sequence in all three strands, the probability of finding conformations with internal loops is small, i.e., 0.06. Internal loops affect the overall orientation of these structures, as measured by the helix-distortion parameters S, K, and U.

The solution conformations of a series of pentapeptides containing the unsaturated reside dehydrophenylalanine have been investigated by infrared, vibrational circular dichroism, electronic CD and NMR spectroscopies. VCD couplets in the NH-stretching region are identified as markers for the left or right-handed helical orientation of NH bonds in these peptides.

Vibrational circular dichroism (VCD) spectra provide information on the solution conformations of chiral molecules. The CH-, OH-, NH-, and C equals O-stretching VCD features are particularly useful conformational probes. Molecules of pharmaceutical interest studied by VCD include biomimetic iron carriers, unsaturated peptides, the anticancer drug taxol and a series of type I antiarrhythmic drugs.© (1993) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Vibrational circular dichroism (VCD) spectra have been recorded in the NH and C equals O stretching regions for a series of tripodal peptides and peptides containing an unsaturated residue, in order to obtain information about solution conformations.© (1992) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Fourier transform ir vibrational circular dichroism (VCD) spectra in the amide I′ region of poly(L-lysine) in D2O solutions have confirmed the existence of three distinct conformational states and an unordered conformational state in this homopolypeptide. Characteristic VCD spectra are presented for the right-handed α-helix, the antiparallel β-sheet, an extended helix conformation previously referred to as the so-called “random coil,” and a completely unordered conformation characterized by the absence of any amide I′ VCD. VCD for the antiparallel β-sheet in solution and the unordered chain conformation are presented for the first time. Each of the four different VCD spectra is unique in appearance and lends weight to the view that VCD has the potential to become a sensitive new probe of the secondary structure of proteins in solution.

Vibrational circular dichroism (VCD) has been used to determine the solution structures of three tripodal peptide molecules. The interchain hydrogen bonding is shown to be oriented in a clockwise direction (from C=0 to NH) by application of the coupled oscillator mechanism in the carbonyl stretching region. The carbonyls of the terminal Boc groups are found to be oriented in a counterclockwise direction. Open chain conformations and dimers are also present in solution and contribute to the VCD spectra with monosignate intensity that is interpreted using the ring current mechanism.