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

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

doi:10.1186/1756-6606-1-16

Fui-Mee Ng, James P Snyder + Show 3 more

Open Access

https://doi.org/10.1186/1756-6606-1-16

Copy DOI

Abstract

BackgroundPhenylethanolamines 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.ResultsLigand 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.ConclusionWe 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.

Highlights

Phenylethanolamines selectively bind to NR2B subunit-containing N-methyl-Daspartate-subtype of ionotropic glutamate receptors and negatively modulate receptor activity
Their relative percentages of α-helix content were estimated to be 26.7 ± 0.4% (n = 5) and 27.7 ± 0.7% (n = 6), Circular dichroism (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
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 dissociation constant (KD) values of 60 ± 18 nM for Buffer-9 (n = 5, Fig. 3A) and 61 ± 34 nM for Buffer-12 (n = 5, Fig. 3B)

Summary

Introduction

Phenylethanolamines selectively bind to NR2B subunit-containing N-methyl-Daspartate-subtype of ionotropic glutamate receptors and negatively modulate receptor activity. 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. N-methyl-D-aspartate (NMDA) receptors are postsynaptic receptors for L-glutamate, the major excitatory neurotransmitter in the central nervous system. They are ligandgated ion channels that are permeable to K+, Na+ and Ca2+. Each NMDA receptor subunit shares a characteristic modular architecture with three transmembrane segments (M1, M3 and M4), a re-entrant loop (initially known as M2) which forms the pore-lining region, an intracellular C-terminal domain, and large extracellular domains [5,10,12]. The remaining non-agonist binding segment preceding M1 is known as the amino-terminal domain (ATD) (Fig. 1A)

Results

Discussion

Conclusion