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


CONCLUSIONS
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.
Ng
Fui-Mee
FM
Department of Pharmacology, National University of Singapore, Singapore. f.m.ng@nus.edu.sg
Geballe
Matthew T
MT
Snyder
James P
JP
Traynelis
Stephen F
SF
Low
Chian-Ming
CM
D000818:Animals; D014150:Antipsychotic Agents / Q000494:pharmacology; D001665:Binding Sites; D002021:Buffers; D004983:Ethanolamines / Q000737:chemistry* / Q000378:metabolism*; D006220:Haloperidol / Q000494:pharmacology; D006639:Histidine; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009842:Oligopeptides; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D010880:Piperidines / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D058849:Protein Refolding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014981:Xenopus
D014150:Antipsychotic Agents; D002021:Buffers; D004983:Ethanolamines; C471213:His-His-His-His-His-His; D008024:Ligands; D050505:Mutant Proteins; C121001:NR2B NMDA receptor; D009842:Oligopeptides; D010880:Piperidines; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; D006639:Histidine; C005332:N-phenylethanolamine; D006220:Haloperidol; C010739:ifenprodil