Successful prediction of the intra- and extracellular loops of four G-protein-coupled receptors

We present results of the restoration of all crystallographically available intra- and extracellular loops of four G-protein-coupled receptors (GPCRs): bovine rhodopsin (bRh), the turkey β-1 adrenergic receptor (β1Ar), and the human β-2 adrenergic (β2Ar) and A2A adenosine (A2Ar) receptors. We use our Protein Local Optimization Program (PLOP), which samples conformational space from first principles to build sets of loop candidates and then discriminates between them using our physics-based, all-atom energy function with implicit solvent. We also discuss a new kind of explicit membrane calculation developed for GPCR loops that interact, either in the native structure or in low-energy false-positive structures, with the membrane, and thus exist in a multiphase environment not previously incorporated in PLOP. Our results demonstrate a significant advance over previous work reported in the literature, and of particular note we are able to accurately restore the extremely long second extracellular loop (ECL2), which is also key for GPCR ligand binding. In the case of β2Ar, accurate ECL2 restoration required seeding a small helix into the loop in the appropriate region, based on alignment with the β1Ar ECL2 loop, and then running loop reconstruction simulations with and without the seeded helix present; simulations containing the helix attain significantly lower total energies than those without the helix, and have rmsds close to the native structure. For β1Ar, the same protocol was used, except the alignment was done to β2Ar. These results represent an encouraging start for the more difficult problem of accurate loop refinement for GPCR homology modeling.