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Mechanisms of antagonism of the GluR2 AMPA receptor: structure and dynamics of the complex of two willardiine antagonists with the glutamate binding domain - PubMed

  • ️Thu Jan 01 2009

Mechanisms of antagonism of the GluR2 AMPA receptor: structure and dynamics of the complex of two willardiine antagonists with the glutamate binding domain

Ahmed H Ahmed et al. Biochemistry. 2009.

Abstract

Ionotropic glutamate receptors mediate the majority of vertebrate excitatory synaptic transmission. The development of selective antagonists for glutamate receptor subtypes is of interest in the treatment of a variety of neurological disorders. This study presents the crystal structure of the binding domain of GluR2 bound to two antagonists (UBP277 and UBP282) that are derivatives of the natural product, willardiine. The antagonists bind to one lobe of the protein with interactions similar to agonists. Interaction with the second lobe differs between the two antagonists, resulting in a different position of the uracil ring and different orientations of the bilobed structure. UBP277 binding produces a stable lobe orientation that is similar to the apo state, but the binding of UBP282 produces the largest hyperextension of the lobes yet reported for an AMPA receptor. The carboxyethyl (UBP277) and carboxybenzyl (UBP282) substituents in the N(3) position keep the lobes separated by a "foot-in-the-door" mechanism and the internal dynamics are minimal compared to the CNQX-bound form of the protein (which makes minimal contacts with one of the two lobes). In contrast to the antagonists CNQX and DNQX, UBP277 and UBP282 produce complexes with higher thermal stability, but affinities that are more than 100-fold lower. These structures support the idea that antagonism is associated with the overall orientation of the lobes rather than with specific interactions, and antagonism can rise either from specific interactions with both lobes ("foot-in-the-door" mechanism) or from the lack of extensive interactions with one of the two lobes.

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Figures

Figure 1
Figure 1

Structures of the binding sites for UBP277 (A) and UBP282 (B) on GluR2 S1S2. In each case, Lobe 1 is shown in cyan and Lobe 2 in green. Interactions between the ligand and protein sidechains are indicated. Additional interactions with the backbone are also made with Lobe 1.

Figure 2
Figure 2

The orientation of different willardiines in the GluR2 S1S2 binding site is illustrated. The residues in Lobe 1 were aligned. The positions of HW (red; 1mqj), BrW (green, 1mqh) and FW (black, 1mqi) are superimposable. However, the positions of IW (blue, 1mqg) varies within the same crystal. IW is the lowest efficacy of the four partial agonists. UBP277 (yellow) and UBP282 (cyan) move even further down the binding pocket than IW and make contact with Lobe 2 through the substituents in the 2 position.

Figure 3
Figure 3

(A) Structures of the antagonists used in the binding study. (B) Inhibition of the binding of [3H]AMPA to GluR2 S1S2 by DNQX, CNQX, UBP277, and UBP282. The IC50 values were: 0.38 ± 0.04 μM, 0.68 ± 0.06 μM, 135 ± 12 μM, 292 ± 26 μM, respectively.

Figure 4
Figure 4

19F NMR spectra of antagonists bound to 5-19F-tryptophan-labeled GluR2 S1S2 as a function of temperature: (A) CNQX, (B) DNQX, (C) UBP277, and (D) UBP282. The signal for W767 is in chemical exchange for the CNQX-bound form but apparently not for S1S2 bound to the other three antagonists. Two additional peaks (marked with asterisks) were consistently seen in spectra of the UBP282-bound form and are consistent with chemical shifts observed in the apo form.

Figure 5
Figure 5

(A) A portion of the 1H,15N-HSQC (TROSY) spectrum at 10°C showing the sidechain tryptophan amides for GluR2 S1S2 bound to a variety of willardiine agonists and antagonists as well as CNQX and DNQX. The signal for W767 varies with the orientation of the two lobes, moving upfield as the lobe opening increases. The amide proton makes a hydrogen bond across the lobe interface with the carbonyl of T707, shown here in the UBP277-S1S2 structure (B). The peak for this correlation is dramatically broadened at this temperature for IW and CNQX, but not the other compounds shown. This broadening is consistent with exchange between multiple relative orientations of the two lobes (10).

Figure 6
Figure 6

(A & B) Sample data from ITC competitive binding studies: 500 μM UBP277 titrated into 12 μM S1S2 bound to glutamate at 10°C. The data were fit using Origin 7 and the equations described by Sigurskjold et al. (33) for competition binding. (C & D) Schematic of the relative enthalpies and entropies of binding of various ligands to S1S2 as determined by competitive binding ITC experiments.

Figure 7
Figure 7

Thermal denaturation of GluR2-S1S2 bound to glutamate, CNQX, DNQX, UBP277 and UBP282. The temperature was increased at a rate of 0.06 min/°C. The data were fit as described by Madden et al. (34).

Figure 8
Figure 8

(A) Ribbon structures of GluR2-S1S2 bound to CNQX (purple, 3b7d, 17), DNQX (brown, 1ftl, 15), UBP277 (yellow), and UBP282 (cyan). The residues in Lobe 1 are aligned to illustrate the different lobe orientations. Using DynDom (41), the lobe opening relative to the glutamate-bound form (1ftj, C protomer, 15) is 15.7° ± 0.5° for CNQX, 16.5° ± 1.1° for DNQX, 19.8° ± 0.6° for UBP277, and 26.7° ± 1.8° for UBP282. (B) Overlay of the binding site for six antagonists showing the multiple sites of interaction. Shown are CNQX, DNQX, UBP277, UBP282 (as in part A), ATPO (orange, 1n0t, 16), and NS1209 (green, 2cmo, 18). As in part A, the residues in Lobe 1 were aligned.

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