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Author Notes:

Correspondence: Chian-Ming Low; phclowcm@nus.edu.sg

Authors' Contributions: FMN carried out the recombinant protein biochemical studies.

MTG carried out and JPS participated in the homology modeling and docking studies.

SFT participated in the functional oocyte studies.

CML conceived of the study, participated in its design and coordination, and drafted the manuscript.

All authors read and approved the final manuscript.

Acknowledgments: We thank Professor Stephen F. Heinemann (Salk Institute) for sharing NR1 and NR2B cDNAs (GenBank accession no. U11418 and U11419).

We thank Professor Xiaodong Cheng and Professor Xing Zhang (Emory University, GA) for valuable advice throughout this study, including hosting C-M.L in their laboratory for 2 months.

We thank Peiqi Lim for her excellent technical assistance and Yoke-Ping Cheong for help in figure preparation.

The authors thank the NUH-NUS Medical Publications Support Unit, Singapore, for assistance in the preparation of this manuscript.

Disclosures: The authors declare that they have no competing interests.


Research Funding:

This work was supported by NIH-NINDS, NARSAD, Michael J Fox Foundation to SFT, Biomedical Research Council grant (R184000119305), Academic Research Funds (R184000114112) and National Medical Research Council grant (0581/2001) to C-M.L.

F-M.N is the recipient of a graduate research scholarship from the National University of Singapore and partially supported by the BMRC grant.

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

Journal Title:

Molecular Brain


Volume 1, Number 16


Type of Work:

Article | Final Publisher PDF


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.

Copyright information:

© 2008 Ng et al; licensee BioMed Central Ltd.

This is an Open Access work distributed under the terms of the Creative Commons Attribution 2.0 Generic License (http://creativecommons.org/licenses/by/2.0/).

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