3D modeling, ligand binding and activation studies of the cloned mouse delta, mu and kappa opioid receptors

Refined 3D models of the transmembrane domains of the cloned delta, mu and kappa opioid receptors belonging to the superfamily of G-protein coupled re ceptors (GPCRs) were constructed from a multiple sequence alignment using t he alpha carbon template of rhodopsin recently reported. Other key steps in the procedure were relaxation of the 3D helix bundle by unconstrained ener gy optimization and assessment of the stability of the structure by perform ing unconstrained molecular dynamics simulations of the energy optimized st ructure. The results were stable ligand-free models of the TM domains of th e three opioid receptors. The ligand-free delta receptor was then used to d evelop a systematic and reliable procedure to identify and assess putative binding sites that would be suitable for similar investigation of the other two receptors and GPCRs in general. To this end, a non-selective, 'univers al' antagonist, naltrexone, and agonist, etorphine, were used as probes. Th ese ligands were first docked in all sites of the model delta opioid recept or which were sterically accessible and to which the protonated amine of th e ligands could be anchored to a complementary proton-accepting residue. Us ing these criteria, nine ligand-receptor complexes with different binding p ockets were identified and refined by energy minimization. The properties o f all these possible ligand-substrate complexes were then examined for cons istency with known experimental results of mutations in both opioid and oth er GPCRs. Using this procedure, the lowest energy agonist-receptor and anta gonist-receptor complexes consistent with these experimental results were i dentified. These complexes were then used to probe the mechanism of recepto r activation by identifying differences in receptor conformation between th e agonist and the antagonist complex during unconstrained dynamics simulati on. The results lent support to a possible activation mechanism of the mous e delta opioid receptor similar to that recently proposed for several other GPCRs. They also allowed the selection of candidate sites for future mutag enesis experiments.