Dynamics of cleft closure of the GluA2 ligand-binding domain in the presence of full and partial agonists revealed by hydrogen-deuterium exchange

Abstract

The majority of excitatory neurotransmission in the CNS is mediated by tetrameric AMPA receptors. Channel activation begins with a series of interactions with an agonist that binds to the cleft between the two lobes of the ligand-binding domain of each subunit. Binding leads to a series of conformational transitions, including the closure of the two lobes of the binding domain around the ligand, culminating in ion channel opening. Although a great deal has been learned from crystal structures, determining the molecular details of channel activation, deactivation, and desensitization requires measures of dynamics and stabilities of hydrogen bonds that stabilize cleft closure. The use of hydrogen-deuterium exchange at low pH provides a measure of the variation of stability of specific hydrogen bonds among agonists of different efficacy. Here, we used NMR measurements of hydrogen-deuterium exchange to determine the stability of hydrogen bonds in the GluA2 (AMPA receptor) ligand-binding domain in the presence of several full and partial agonists. The results suggest that the stabilization of hydrogen bonds between the two lobes of the binding domain is weaker for partial than for full agonists, and efficacy is correlated with the stability of these hydrogen bonds. The closure of the lobes around the agonists leads to a destabilization of the hydrogen bonding in another portion of the lobe interface, and removing an electrostatic interaction in Lobe 2 can relieve the strain. These results provide new details of transitions in the binding domain that are associated with channel activation and desensitization.

Keywords: Glutamate Receptors; Hydrogen-Deuterium Exchange; Ionotropic Glutamate Receptors (AMPA, NMDA); Membrane Proteins; NMR; Neurotransmitter Receptors; Willardiine.

FIGURE 1.

Schematic illustrating the three steps of binding of full agonist to the GluA2 LBD (16): 1) binding of agonist to Lobe 1, 2) closure of the lobes and binding to Lobe 2, and 3) formation of H-bonds across the lobe interface. In this and all subsequent figures, Lobe 1 is colored blue, and Lobe 2 is colored green. The apo form (upper left) is the A-protomer of the apo structure of the GluA2 LBD (Protein Data Bank code 1FTO) (2). Step 1 (upper right) is a superposition (alignment of backbones of Lobe 1 for each structure) of the glutamate-bound form (code 3DP6) (20) with the apo form, showing the protein of the apo form and only the ligand (glutamate) for the glutamate-bound form. Step 2 (lower left) is a superposition of the glutamate-bound form (code 3DP6) with the IW-bound form (code 1MQG) (10), showing only the protein of the IW-bound form and only the ligand (glutamate) for the glutamate-bound form. Step 3 (lower right) shows the glutamate-bound form (code 3DP6).

FIGURE 2.

A, HD exchange at Thr-480 for several agonists. B, structure of the glutamate-bound GluA2 LBD (Protein Data Bank code 3DP6) (20) showing interactions with Lobe 1 (Step 1).

FIGURE 3.

A, HD exchange at Thr-655 for several agonists. The signal at Thr-655 for NW is lost after the third spectrum, so only two data points are shown. In this case, a line is shown only to draw attention to the point rather than to suggest an accurate exchange rate. B, pH-dependent ionization state of NW. C–E, H-bonds of AMPA (Protein Data Bank code 1FTM) (2), glutamate (code 3DP6) (20), kainate (code 1FW0) (2), and NW (code 3RTW) (13), respectively, with residues in Lobe 2 (Step 2).

FIGURE 4.

A–C, HD exchange at the backbone amide of Tyr-450, the side chain of Trp-767, and the side chain of Trp-766, respectively. D and E, H-bonds formed across the lobe interface near the binding site in the NW-bound form (Step 3; Protein Data Bank code 3RTW) (13). The flipped form is shown in D, and the unflipped form is shown in E. F, H-bonds involving tryptophan side chains formed near the disulfide bond.

FIGURE 5.

A and B, HD exchange for the E713T mutant and wild-type LBDs at the side chain of Trp-766 and the backbone of Tyr-450, respectively. C, comparison of the thermal stability of the E713T mutant relative to the wild-type GluA2 LBD. D, binding of [³H]AMPA to the wild-type and E713T mutant GluA2 LBDs. Data were fit with a model that assumes one binding site per receptor, with IC₅₀ values of 5.3 μm for the wild-type GluA2 LBD and 1.7 μm for the E713T mutant. E, residues showing differences in HD exchange in the E713T versus wild-type LBD. Those with a faster exchange rate in the mutant are shown in red, and those with a slower exchange rate in the mutant are shown in blue. In both E and F, Glu-713 is shown in magenta. F, changes in chemical shift (31) in the E713T mutant versus wild-type LBD when bound to glutamate. Yellow represents a shift change of ∼0.05 ppm, and red represents a shift change >0.1 ppm. G, points of contact between Lobes 1 and 2 and the interaction between Glu-713 and Lys-722 are shown. H, hydrophobic cores of Lobes 1 and 2 as defined previously (32). The spheres are the positions of the methyl groups. Note that Glu-713 is near the axis of rotation for lobe closure and defines a point of contact between Core B and the β-sheet.