The Orientations of Proteins in Membranes (OPM) database is a curated web resource that provides spatial positions of membrane-bound peptides and proteins of known three-dimensional structure in the lipid bilayer, together with their structural classification, topology and intracellular localization. OPM currently contains more than 1200 transmembrane and peripheral proteins and peptides from approximately 350 organisms that represent approximately 3800 Protein Data Bank entries. Proteins are classified into classes, superfamilies and families and assigned to 21 distinct membrane types. Spatial positions of proteins with respect to the lipid bilayer are optimized by the PPM 2.0 method that accounts for the hydrophobic, hydrogen bonding and electrostatic interactions of the proteins with the anisotropic water-lipid environment described by the dielectric constant and hydrogen-bonding profiles. The OPM database is freely accessible at http://opm.phar.umich.edu. Data can be sorted, searched or retrieved using the hierarchical classification, source organism, localization in different types of membranes. The database offers downloadable coordinates of proteins and peptides with membrane boundaries. A gallery of protein images and several visualization tools are provided. The database is supplemented by the PPM server (http://opm.phar.umich.edu/server.php) which can be used for calculating spatial positions in membranes of newly determined proteins structures or theoretical models.

http://opm.phar.umich.edu.

The endogenous opioid pentapeptides [Met5]enkephalin (H-TyrGly-Gly-Phe-Met-OH) and [Leu5]enkephalin (H-Tyr-Gly-GlyPhe-Leu-OH) have been shown to interact with several classes of opioid receptors (1-3) that may mediate different physiological responses. Elucidation of the roles of the individual receptor classes has been hampered by the general lack of enkephalin analogs with a high degree of selectivity for a single receptor type. The vast majority of analogs crossreact extensively with the different receptors, making it difficult to define receptor roles. This situation has been in part ameliorated by recent reports of an enkephalin analog highly selective for the ,At opioid receptor (4-6) and a nonpeptide opiate with high K receptor selectivity (7). However, analogs with corresponding selectivity for the 8 opioid receptor have not been demonstrated.One approach for the design of more selective analogs involves the incorporation of conformational restrictions. The native enkephalins, like most small, linear peptides, possess considerable conformational flexibility and by virtue of this flexibility can attain the presumably different conformational features required for interaction with different classes of opioid receptors. In principle, appropriate restriction of this flexibility can lead to analogs able to assume the conformation required to interact favorably with only one class of receptor. One method for effecting conformational restrictions is via cyclization of the peptide that constrains the resulting analog to assume a compact topography. Several active, cyclic enkephalin analogs have been reported, all of which are cyclized by either side chain to carboxyl terminus (8,9) It has previously been shown that, in aqueous solution, the tocin ring portion of Pen-containing oxytocin analogs is conformationally restricted, whereas the tocin ring of oxytocin itself is quite flexible (13-16). This difference arises from the rigidifying effect of gem-dialkyl substituents in medium-sized rings and suggests that the 8 (Fig. 1) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

A new computational approach has been developed to determine the spatial arrangement of proteins in membranes by minimizing their transfer energies from water to the lipid bilayer. The membrane hydrocarbon core was approximated as a planar slab of adjustable thickness with decadiene-like interior and interfacial polarity profiles derived from published EPR studies. Applicability and accuracy of the method was verified for a set of 24 transmembrane proteins whose orientations in membranes have been studied by spin-labeling, chemical modification, fluorescence, ATR FTIR, NMR, cryo-microscopy, and neutron diffraction. Subsequently, the optimal rotational and translational positions were calculated for 109 transmembrane, five integral monotopic and 27 peripheral protein complexes with known 3D structures. This method can reliably distinguish transmembrane and integral monotopic proteins from water-soluble proteins based on their transfer energies and membrane penetration depths. The accuracies of calculated hydrophobic thicknesses and tilt angles were ;1 Å and 2°, respectively, judging from their deviations in different crystal forms of the same proteins. The hydrophobic thicknesses of transmembrane proteins ranged from 21.1 to 43.8 Å depending on the type of biological membrane, while their tilt angles with respect to the bilayer normal varied from zero in symmetric complexes to 26°in asymmetric structures. Calculated hydrophobic boundaries of proteins are located ;5 Å lower than lipid phosphates and correspond to the zero membrane depth parameter of spin-labeled residues. Coordinates of all studied proteins with their membrane boundaries can be found in the Orientations of Proteins in Membranes (OPM) database: http://opm.phar.umich.edu/.

Three-dimensional structures of the transmembrane, seven alpha-helical domains and extracellular loops of delta, mu, and kappa opioid receptors, were calculated using the distance geometry algorithm, with hydrogen bonding constraints based on the previously developed general model of the transmembrane alpha-bundle for rhodopsin-like G-protein coupled receptors (Biophys. J. 1997. 70:1963). Each calculated opioid receptor structure has an extensive network of interhelical hydrogen bonds and a ligand-binding crevice that is partially covered by a beta-hairpin formed by the second extracellular loop. The binding cavities consist of an inner "conserved region" composed of 18 residues that are identical in delta, mu, and kappa opioid receptors, and a peripheral "variable region," composed of 19 residues that are different in delta, mu, and kappa subtypes and are responsible for the subtype specificity of various ligands. Sixteen delta-, mu-, or kappa-selective, conformationally constrained peptide and nonpeptide opioid agonists and antagonists and affinity labels were fit into the binding pockets of the opioid receptors. All ligands considered have a similar spatial arrangement in the receptors, with the tyramine moiety of alkaloids or Tyr1 of opioid peptides interacting with conserved residues in the bottom of the pocket and the tyramine N+ and OH groups forming ionic interactions or H-bonds with a conserved aspartate from helix III and a conserved histidine from helix VI, respectively. The central, conformationally constrained fragments of the opioids (the disulfide-bridged cycles of the peptides and various ring structures in the nonpeptide ligands) are oriented approximately perpendicular to the tyramine and directed toward the extracellular surface. The results obtained are qualitatively consistent with ligand affinities, cross-linking studies, and mutagenesis data.