br Ligand binding free energies In
Ligand-binding free energies In order to compute the absolute free Pralatrexate associated with binding a ligand to a flexible iGluR LBD, a theory that accounts for all the principal thermodynamic contributions from ligand docking and LBD closure had to be developed. This was accomplished using an approach involving umbrella sampling PMF and free energy perturbation (FEP) calculations . FEP calculations [42,43] consist of evaluating changes in free energy during step-wise (non-physical) alchemical transformations. The PMF-based approach, which follows from a rigorous statistical mechanical formulation of noncovalent binding, was applied to a panel of nine ligands that included full and partial agonists and antagonists of GluA2. The results agreed well with experimentally measured apparent affinities to the isolated LBD. The different thermodynamic contributions associated with ligand docking and LBD closure for the nine ligands were dissected. An analysis of accessible LBD conformations transposed onto an intact GluA2 receptor revealed that the relative dynamics between certain diagonally positioned subunits may explain the action of partial agonists. Umbrella sampling and metadynamics  were also used to compute binding free energies of AMPA and 2-BnTetAMPA, a photoswitchable agonist, to the GluA2 LBD . In a separate study, the standard binding free energies of five agonists and antagonists of GluA2 (ACPA, AMPA, CNQX, DNQX, and glutamate) were computed  using FEP and thermodynamic integration (TI) [47,48], a process related to FEP, in conjunction with the double decoupling method (DDM) [, , ], in which interactions between the ligand and its environment are progressively switched off. The simulation systems were set up such that transformations of the ligand in the binding site occurred simultaneously with transformations of the ligand in bulk solvent; this allowed calculations to be carried out on ligands with net charge. The confine-and-release strategy , a hybrid of FEP and PMF calculations, was employed to address free energies linked to conformational changes in the LBD that occur when a ligand is introduced into the binding site. Using this approach, and including the calculated free energy of LBD closure from Lau and Roux , standard binding free energies in good agreement with both experimentally measured values and calculated values were obtained.
LBD dimers Kainate receptors require both anions and cations for activation, and these ions also play roles in regulating desensitization [53,54]. Relative binding free energy calculations using TI were carried out to examine the affinity of different monovalent cations in the GluK1 (named GluR5 at the time) LBD dimer interface . The calculated affinities were found to generally correlate with experimental apparent affinities measured in functional assays. Computed PMFs show that the energetic barrier for translocating an anion into the interface is lowered by bound cations. Relative binding free energy calculations were also performed for Na+ and Li+ in the GluK2 homodimer and the GluK2-GluK5 heterodimer to examine the relationship between packing of the dimer interface and entry into desensitization . Li+ was predicted to bind tightly in the GluK2-GluK5 dimer interface and, consequently, slow desensitization. The predictions were validated using electrophysiological recordings. Steered MD (SMD) simulations , a nonequilibrium process in which the system is driven to a target conformation using applied forces, were used to calculate the work required to disrupt LBD dimers from kainate and AMPA receptors . The results were found to correlate well with measured resistance to desensitization, as reported in the literature. An inconsistency in prior interpretations of experimental results regarding GluA2 desensitization [8,59] was at least partly resolved by this study. Specifically, a disulfide-crosslinked GluA2 LBD dimer formed by the K514C mutation was found, surprisingly, to require a similar amount of work as WT GluA2 to adopt a conformation consistent with entry into desensitization.