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

Ng FM, Geballe MT, Snyder JP, Traynelis SF, Low CM - Mol Brain (2008)

Bottom Line: Molecular modeling of ATD2B as a clam-shell-like structure places these critical residues near a putative ligand binding site.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, National University of Singapore, Singapore. f.m.ng@nus.edu.sg

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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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.
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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.

Mentions: CD spectra of the Buffers -9 (Fig. 2A) and -12 refolded (Fig. 2B) 6 × His-ATD2B proteins were obtained in the presence of 5 μM ifenprodil and 0.5 μM RO25,6981. Ifenprodil and RO25,6981 in the concentration ranges 0.01–5 μM and 0.002–0.5 μM, respectively, diminished the θ over the wavelengths in the region 210–250 nm. We interpret the concentration-dependent shifts of magnitude at wavelength 220.0 nm ellipticity to suggest that ifenprodil binds to refolded soluble 6 × His-ATD2B proteins with KD values of 60 ± 18 nM for Buffer-9 (n = 5, Fig. 3A) and 61 ± 34 nM for Buffer-12 (n = 5, Fig. 3B). The concentration-dependent changes in the CD spectrum induced by RO25,6981 (218.9 nm) upon interacting with the 6 × His-ATD2B proteins suggest that the ligand binds with KD values of 7 ± 3 nM (Buffer-9), 4 ± 1 nM (Buffer-12) (n = 5, Figs. 3C, D) and 4 ± 2 nM (Buffer-13). Figure 4A summarizes the binding properties of 6 × His-ATD2B to ifenprodil and RO25,6981 between three different protein refolding buffers. In addition, in the presence of saturating concentration of 5 μM ifenprodil, RO25,6891 could displace ifenprodil and bind to 6 × His-ATD2B with comparable affinities in the absence of ifenprodil: Buffer -9 (4 ± 1 nM), Buffer -12 (2 ± 1 nM) and Buffer -13 (3 ± 1 nM).


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

Ng FM, Geballe MT, Snyder JP, Traynelis SF, Low CM - Mol Brain (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2603005&req=5

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.
Mentions: CD spectra of the Buffers -9 (Fig. 2A) and -12 refolded (Fig. 2B) 6 × His-ATD2B proteins were obtained in the presence of 5 μM ifenprodil and 0.5 μM RO25,6981. Ifenprodil and RO25,6981 in the concentration ranges 0.01–5 μM and 0.002–0.5 μM, respectively, diminished the θ over the wavelengths in the region 210–250 nm. We interpret the concentration-dependent shifts of magnitude at wavelength 220.0 nm ellipticity to suggest that ifenprodil binds to refolded soluble 6 × His-ATD2B proteins with KD values of 60 ± 18 nM for Buffer-9 (n = 5, Fig. 3A) and 61 ± 34 nM for Buffer-12 (n = 5, Fig. 3B). The concentration-dependent changes in the CD spectrum induced by RO25,6981 (218.9 nm) upon interacting with the 6 × His-ATD2B proteins suggest that the ligand binds with KD values of 7 ± 3 nM (Buffer-9), 4 ± 1 nM (Buffer-12) (n = 5, Figs. 3C, D) and 4 ± 2 nM (Buffer-13). Figure 4A summarizes the binding properties of 6 × His-ATD2B to ifenprodil and RO25,6981 between three different protein refolding buffers. In addition, in the presence of saturating concentration of 5 μM ifenprodil, RO25,6891 could displace ifenprodil and bind to 6 × His-ATD2B with comparable affinities in the absence of ifenprodil: Buffer -9 (4 ± 1 nM), Buffer -12 (2 ± 1 nM) and Buffer -13 (3 ± 1 nM).

Bottom Line: Molecular modeling of ATD2B as a clam-shell-like structure places these critical residues near a putative ligand binding site.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, National University of Singapore, Singapore. f.m.ng@nus.edu.sg

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.

Show MeSH
Related in: MedlinePlus