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 ß-1 adrenergic receptor (ß1Ar), and the human ß-2 adrenergic (ß2Ar) 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 ß2Ar, accurate ECL2 restoration required seeding a small helix into the loop in the appropriate region, based on alignment with the ß1Ar 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 ß1Ar, the same protocol was used, except the alignment was done to ß2Ar. These results represent an encouraging start for the more difficult problem of accurate loop refinement for GPCR homology modeling.

Loop prediction for a GPCR homology model: algorithms and results.

We present loop structure prediction results of the intracellular and extracellular loops of four G-protein-coupled receptors (GPCRs): bovine rhodopsin (bRh), the turkey ß1-adrenergic (ß1Ar), the human ß2-adrenergic (ß2Ar) and the human A2a adenosine receptor (A2Ar) in perturbed environments. We used the protein local optimization program, which builds thousands of loop candidates by sampling rotamer states of the loops' constituent amino acids. The candidate loops are discriminated between with our physics-based, all-atom energy function, which is based on the OPLS force field with implicit solvent and several correction terms. For relevant cases, explicit membrane molecules are included to simulate the effect of the membrane on loop structure. We also discuss a new sampling algorithm that divides phase space into different regions, allowing more thorough sampling of long loops that greatly improves results. In the first half of the paper, loop prediction is done with the GPCRs' transmembrane domains fixed in their crystallographic positions, while the loops are built one-by-one. Side chains near the loops are also in non-native conformations. The second half describes a full homology model of ß2Ar using ß1Ar as a template. No information about the crystal structure of ß2Ar was used to build this homology model. We are able to capture the architecture of short loops and the very long second extracellular loop, which is key for ligand binding. We believe this the first successful example of an RMSD validated, physics-based loop prediction in the context of a GPCR homology model.

Localized orbital corrections applied to thermochemical errors in density functional theory: The role of basis set and application to molecular reactions.

This paper is a logical continuation of the 22 parameter, localized orbital correction (LOC) methodology that we developed in previous papers [R. A. Friesner et al., J. Chem. Phys. 125, 124107 (2006); E. H. Knoll and R. A. Friesner, J. Phys. Chem. B 110, 18787 (2006).] This methodology allows one to redress systematic density functional theory (DFT) errors, rooted in DFT's inherent inability to accurately describe nondynamical correlation. Variants of the LOC scheme, in conjunction with B3LYP (denoted as B3LYP-LOC), were previously applied to enthalpies of formation, ionization potentials, and electron affinities and showed impressive reduction in the errors. In this paper, we demonstrate for the first time that the B3LYP-LOC scheme is robust across different basis sets [6-31G( *), 6-311++G(3df,3pd), cc-pVTZ, and aug-cc-pVTZ] and reaction types (atomization reactions and molecular reactions). For example, for a test set of 70 molecular reactions, the LOC scheme reduces their mean unsigned error from 4.7 kcal/mol [obtained with B3LYP/6-311++G(3df,3pd)] to 0.8 kcal/mol. We also verified whether the LOC methodology would be equally successful if applied to the promising M05-2X functional. We conclude that although M05-2X produces better reaction enthalpies than B3LYP, the LOC scheme does not combine nearly as successfully with M05-2X than with B3LYP. A brief analysis of another functional, M06-2X, reveals that it is more accurate than M05-2X but its combination with LOC still cannot compete in accuracy with B3LYP-LOC. Indeed, B3LYP-LOC remains the best method of computing reaction enthalpies.

Docking and free energy perturbation studies of ligand binding in the kappa opioid receptor.

The kappa opioid receptor (KOR) is an important target for pain and depression therapeutics that lack harmful and addictive qualities of existing medications. We present a model for the binding of morphinan ligands and JDTic to the JDTic/KOR crystal structure based on an atomic level description of the water structure within its active site. The model contains two key interaction motifs that are supported by experimental evidence. The first is the formation of a salt bridge between the ligand and Asp 138(3.32) in transmembrane domain (TM) 3. The second is the stabilization by the ligand of two high energy, isolated, and ice-like waters near TM5 and TM6. This model is incorporated via energetic terms into a new empirical scoring function, WScore, designed to assess interactions between ligands and localized water in a binding site. Pairing WScore with the docking program Glide discriminates known active KOR ligands from large sets of decoy molecules much better than Glide's older generation scoring functions, SP and XP. We also use rigorous free energy perturbation calculations to provide evidence for the proposed mechanism of interaction between ligands and KOR. The molecular description of ligand binding in KOR should provide a good starting point for future drug discovery efforts for this receptor.

The protein local optimization program and G-protein-coupled receptors: loop restoration and applications to homology modeling.

The protein local optimization program (PLOP) uses sophisticated sampling algorithms and a highly refined physics-based energy function to restore loops within a protein structure. In this chapter, we highlight some of the recent successes we have had with PLOP restoring long loops in their native environment as well as the intra- and extracellular loops of four G-protein-coupled receptors. This includes the very long second extracellular loops of bovine rhodopsin and the turkey ß1- and human ß2-adrenergic receptors. We then provide an extremely detailed description of PLOP's algorithms, as well as a sample file and explicit keywords so that a new user can successfully run PLOP.

Computational methods for high resolution prediction and refinement of protein structures.

We review advances in implicit solvation and sampling algorithms which have resulted in enhanced capabilities in predicting and refining localized protein structures (e.g. loop regions) to high resolution. Improvements in the generalized Born model and hydrophobicity term yield significantly more accurate energetics; specialized sampling algorithms allow complex local structures, such as a loop-helix-loop region, to be reliably predicted. A novel penalty term is added for loops containing patterns of dihedrals seldom found in experimental structures. We show prediction of diverse sets of large loops, in the native backbone environment, to subångström accuracy. The methodology offers the promise of addressing the refinement problem in homology modeling if an approach can be devised to handle delocalized errors in the structure.

Prediction of Long Loops with Embedded Secondary Structure using the Protein Local Optimization Program.

Robust homology modeling to atomic-level accuracy requires in the general case successful prediction of protein loops containing small segments of secondary structure. Further, as loop prediction advances to success with larger loops, the exclusion of loops containing secondary structure becomes awkward. Here, we extend the applicability of the Protein Local Optimization Program (PLOP) to loops up to 17 residues in length that contain either helical or hairpin segments. In general, PLOP hierarchically samples conformational space and ranks candidate loops with a high-quality molecular mechanics force field. For loops identified to possess α-helical segments, we employ an alternative dihedral library composed of (ϕ,ψ) angles commonly found in helices. The alternative library is searched over a user-specified range of residues that define the helical bounds. The source of these helical bounds can be from popular secondary structure prediction software or from analysis of past loop predictions where a propensity to form a helix is observed. Due to the maturity of our energy model, the lowest energy loop across all experiments can be selected with an accuracy of sub-Ångström RMSD in 80% of cases, 1.0 to 1.5 Å RMSD in 14% of cases, and poorer than 1.5 Å RMSD in 6% of cases. The effectiveness of our current methods in predicting hairpin-containing loops is explored with hairpins up to 13 residues in length and again reaching an accuracy of sub-Ångström RMSD in 83% of cases, 1.0 to 1.5 Å RMSD in 10% of cases, and poorer than 1.5 Å RMSD in 7% of cases. Finally, we explore the effect of an imprecise surrounding environment, in which side chains, but not the backbone, are initially in perturbed geometries. In these cases, loops perturbed to 3Å RMSD from the native environment were restored to their native conformation with sub-Ångström RMSD.

Localized orbital corrections for the calculation of barrier heights in density functional theory.

This work describes the extension of a previously reported empirical localized orbital correction model for density functional theory (DFT-LOC) for atomization energies, ionization potentials, electron affinities, and reaction enthalpies to the correction of barrier heights for chemical reactions of various types including cycloadditions, cycloreversions, dipolar cycloadditions, S(N)2's, carbon radical reactions, hydrogen radical reactions, sigmatropic shifts, and electrocyclizations. The B3LYP localized orbital correction version of the model (B3LYP-LOC) reduces the number of outliers and overall mean unsigned error (MUE) vs. experiment or ab initio values from 3.2 to 1.3 kcal/mole for barrier heights and from 5.1 to 1.1 kcal/mole for reaction enthalpies versus B3LYP. Furthermore, the new model has essentially zero additional computational cost beyond standard DFT calculations. Although the model is heuristic and is based on multiple linear regression to experimental or ab initio data, each of the parameters is justified on chemical grounds and provides insight into the fundamental limitations of DFT, most importantly the failure of current DFT methods to accurately account for nondynamical electron correlation.

Origins of stereoselectivity in the oxido-alkylidenation of alkynes.

A mild, convenient oxido-alkylidenation of alkynes is described. The three-step sequence involves the 1,3-dipolar cycloaddition of a nitrone and an alkynoate, oxidation of the resulting isoxazoline, and stereoselective extrusion of nitrosomethane. Quantum mechanical calculations identified the interactions of R3 with the oxidant and the preferred conformation of a diradical intermediate as major factors controlling the stereoselectivity of the oxidation and torquoselectivity of the extrusion.