The hidden energetics of ligand binding and activation in a glutamate receptor

Abstract

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate most excitatory synaptic transmission in the central nervous system. The free energy of neurotransmitter binding to the ligand-binding domains (LBDs) of iGluRs is converted into useful work to drive receptor activation. We have computed the principal thermodynamic contributions from ligand docking and ligand-induced closure of LBDs for nine ligands of GluA2 using all-atom molecular dynamics free energy simulations. We have validated the results by comparison with experimentally measured apparent affinities to the isolated LBD. Features in the free energy landscapes that govern closure of LBDs are key determinants of binding free energies. An analysis of accessible LBD conformations transposed into the context of an intact GluA2 receptor revealed that the relative displacement of specific diagonal subunits in the tetrameric structure may be key to the action of partial agonists.

Figure 1

Ligands of GluA2. The full agonists (top row) are glutamate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and (S)-2-amino-3-(3-carboxy-5-methylisoxazol-4-yl)propionic acid (ACPA). The partial agonists (middle row) are kainate, (S)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isothiazolyl)propionic acid (thio-ATPA), and (S)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]-4-isoxazolyl)propionic acid (4-AHCP). The antagonists (bottom row) are (S)-2-Amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid (ATPO), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and 6,7-dinitroquinoxaline-2,3 dione (DNQX). Crystal structures of each of these ligands in complex with the GluA2 LBD are used for our molecular models,,,–.

Figure 2

LBD conformational distributions. (a) The free energy landscapes governing LBD closure for the holo and apo proteins calculated from all-atom umbrella sampling MD simulations with explicit solvent. Each contour line corresponds to 1 kcal mol⁻¹, with the darker colors indicating more favorable conformations. The free energy minimum associated with the most closed conformation is for AMPA (ξ₁, ξ₂) = (9.2 Å, 8.4 Å). The conformation used for the ligand-docking simulations is (14.4 Å, 13.7 Å). These locations are indicated by the dotted lines in each panel for reference. (b) The 2D order parameter (ξ₁, ξ₂) describing closure of the GluA2 LBD. Each distance (dashed line) is measured between the center-of-mass (COM) of the residues whose atoms are shown as spheres. The crystal structure of the open, apo LBD (1FTO) is shown.

Figure 3

Comparison of calculated free energy contributions with experimentally measured effective ligand-binding affinities to the isolated GluA2 LBD. (a) Calculated $\mathrm{\Delta }{G}_{\text{dock}}^{(◦)}$. (b) Calculated ΔG_close. (c) Calculated $\mathrm{\Delta }{G}_{\text{bind}}^{(◦)}=\mathrm{\Delta }{G}_{\text{dock}}^{(◦)}+\mathrm{\Delta }{G}_{\text{close}}$. In each plot, the solid line, which has slope = 1, indicates perfect agreement between the calculated and experimental values. The dashed lines are linear regression fits to the data, and their slopes and correlation coefficients are reported. Each ligand is marked numerically in increasing order from the highest experimentally measured affinity to the lowest affinity (see Supplementary Table 1): 1 = AMPA, 2 = ACPA, 3 = 4-AHCP, 4 = CNQX, 5 = glutamate, 6 = thio-ATPA, 7 = DNQX, 8 = kainate, and 9 = ATPO.

Figure 4

LBD conformational distributions in the context of an intact receptor. (a, left) Superposition of LBD conformations spanning the free energy landscapes onto the crystal structure of the intact GluA2 receptor (gray²; the ATD is not shown). The LBDs were superimposed only in Lobe 1. (a, right) Labeling of the four LBDs in an intact iGluR, as viewed from above. The LBDs assemble as a pair of dimers, where A–D is one dimer and B–C is the other. (b) R.m.s.d. distributions of LBD conformations relative to the intact receptor. The r.m.s.d. was measured in regions in Lobe 2 (see Online Methods). The solid line indicates the average r.m.s.d. (see Supplementary Table 5). The dashed line indicates the r.m.s.d. measured from the isolated LBD–ligand crystal structure.

Figure 5

Inter-LBD distance distributions. LBD conformations were superimposed onto the intact GluA2 structure (see Fig. 4), and the pairwise distances were measured between regions in Lobe 2 (see Online Methods). The apo LBD is gray, the LBD–antagonist complexes are blue, and the LBD–full agonist complexes are red. The LBD–partial agonist complexes for (a) kainate, (b) 4-AHCP, and (c) thio-ATPA are green. See Supplementary Table 5 for statistics and the distances measured using the isolated LBD–ligand crystal structures.