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. 2008 Nov 18:1:16.
doi: 10.1186/1756-6606-1-16.

Structural insights into phenylethanolamines high-affinity binding site in NR2B from binding and molecular modeling studies

Fui-Mee Ng  1 , Matthew T Geballe, James P Snyder, Stephen F Traynelis, Chian-Ming Low
Affiliations

Structural insights into phenylethanolamines high-affinity binding site in NR2B from binding and molecular modeling studies

Fui-Mee Ng et al. Mol Brain. .

Abstract

Background: Phenylethanolamines selectively bind to NR2B subunit-containing N-methyl-D-aspartate-subtype of ionotropic glutamate receptors and negatively modulate receptor activity. To investigate the structural and functional properties of the ifenprodil binding domain on the NR2B protein, we have purified a soluble recombinant rat NR2B protein fragment comprising the first ~400 amino acid amino-terminal domain (ATD2B) expressed in E. coli. Spectral measurements on refolded ATD2B protein demonstrated specific binding to ifenprodil. We have used site-directed mutagenesis, circular dichroism spectroscopy and molecular modeling to obtain structural information on the interactions between critical amino acid residues and ifenprodil of our soluble refolded ATD2B proteins. Ligand-induced changes in protein structure were inferred from changes in the circular dichroism spectrum, and the concentration dependence of these changes was used to determine binding constants for ifenprodil and its analogues.

Results: Ligand binding of ifenprodil, RO25,6981 and haloperidol on soluble recombinant ATD2B determined from circular dichroism spectroscopy yielded low-to-high micromolar equilibrium constants which concurred with functional IC₅₀ measurement determined in heterologously expressed NR1/NR2B receptors in Xenopus oocytes. Amino acid residue substitutions of Asp101, Ile150 and Phe176 with alanine residue within the ATD2B protein altered the recombinant protein dissociation constants for ifenprodil, mirroring the pattern of their functional phenotypes. Molecular modeling of ATD2B as a clam-shell-like structure places these critical residues near a putative ligand binding site.

Conclusion: We report for the first time biochemical measurements show that the functional measurements actually reflect binding to the ATD of NR2B subunit. Insights gained from this study help advance the theory that ifenprodil is a ligand for the ATD of NR2B subunit.

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Figures

Figure 1
Figure 1
Modular organization of NR2B subunit of NMDA receptor. A. Diagram shows the amino-terminal domain (ATD), the two lobes (S1–S2) comprising the glutamate binding domain, the three transmembrane domains (M1, M3 and M4), the re-entrant loop and the intracellular cytoplasmic terminus (-COOH). ATD has been proposed to harbor the binding site for phenylethanolamines such as ifenprodil. B. Chemical structures of ifenprodil, RO25,6981 and haloperidol – ligands that bind to and negatively modulate the current flux through NR2B subunit-containing NMDA receptors.
Figure 2
Figure 2
Representative CD spectra of soluble 6 × His-ATD2B proteins in the presence and absence of ligands at 25°C. A. Buffer -9 refolded protein; B. Buffer -12 refolded protein; Solid line, protein in absence of ligand; Dotted line, in presence of 5 μM ifenprodil; Dashed line, in presence of 0.5 μM RO25,6981; Dashed-dotted line, 4 M Gdn.HCl-denatured protein. Y-axis represents the molecular ellipticity. X-axis represents the range of wavelengths analyzed.
Figure 3
Figure 3
Ifenprodil and RO25,6981 titration curves of soluble 6 × His-ATD2B proteins. A. Buffer -9 refolded protein in the presence of ifenprodil; B. Buffer -12 refolded protein in the presence of ifenprodil; C. Buffer -9 refolded protein in the presence of RO25,6981; D Buffer -12 refolded protein in the presence of RO25,6981. The results are the average of five experiments using proteins obtained from at least two independent batches of bacteria induction. Error bars represent S.E.
Figure 4
Figure 4
Bar chart showing the relative affinities of ifenprodil and RO25,6981 towards the three buffers' refolded 6 × His-ATD2B proteins. A. Results are the average of five experiments using proteins obtained from at least two independent batches of bacteria induction. Error bars represent S.E. Ratios of KD(ifenprodil) to KD(RO25,6981) are 9 (Buffer-9), 15 (Buffer-12) and 24 (Buffer-13), respectively. B. Composite antagonists' inhibition curves are shown for NR1 coexpressed with wild-type NR2B in Xenopus oocytes. Error bars represent ± SEM. C. The Log IC50 for RO25,6981, ifenprodil and haloperidol plotted against their respective Log KD values obtained from dose-titration using CD. A linear regression fit revealed good correlation (R = 0.96) between the mean Log IC50 and Log KD values for each ligand. The mean of the sum of individual log value obtained from each fitted dose-inhibition plot for each ligand and SEM are plotted. Mean KD and IC50 values are given in Table 1.
Figure 5
Figure 5
Ifenprodil titration curve for Buffer -9 refolded wild type and mutant proteins. A. Average dose-titration plots showing reduced affinity of ifenprodil to the F176A mutant protein (n = 5) as compared to the wild type protein (n = 5). The mutant protein harboring double mutations D101A/F176A (n = 5) (open circle) showed further reduction in affinity compared to the single mutant, F176A (filled circle). B. Mutation at residue Asp113, which was identified outside the putative ifenprodil binding pocket by our homology model, did not affect the binding affinity significantly (n = 5, P > 0.4). The KD values for different mutant proteins are listed in Table 2. Each data point is the average value obtained from five experiments using proteins obtained from at least two independent batches of bacteria induction. C. Mean-fitted KD values determined for WT protein and proteins containing the respective mutations. Paired Student t-Tests were performed to evaluate the difference between mean of KD(mutant) and KD(WT), * P < 0.05.
Figure 6
Figure 6
Homology model of ATD of NR2B subunit. A. Ribbon representation of the amino-terminal domain of NR2B was constructed as described under Materials and Methods using mGluR1 crystal structure (1 EWK) as template [36]. Model resembles the clam-shell configuration as seen in the bacterial periplasmic leucine-isoleucine-valine binding protein (LIVBP) (2 LIV). Critical residues (Asp101, Ile150, Phe176) that reduced ifenprodil sensitivity 60-fold and more, when mutated, as compared to wild-type NMDA receptors [23] are shown as red sticks using Rasmol. A mutation (D113A) outside the putative ifenprodil binding pocket is identified by blue sticks. B. Putative ifenprodil binding pocket highlighting the three critical amino acid residues that play critical role in interacting with ligand and the inhibition sensitivity of NR2B-containing NMDA receptors. C. Ifenprodil-docked into ATD2B putative binding pocket model. The relative orientations of side chains of Asp101 (top red residue in red sticks), Ile150 and Phe176 towards the putative centre cleft suggest plausible ligand-protein interactions with various functional groups on ifenprodil (see Discussion).
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