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Nature
2011 Jun 15;4757355:249-53. doi: 10.1038/nature10180.
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Subunit arrangement and phenylethanolamine binding in GluN1/GluN2B NMDA receptors.
Karakas E
,
Simorowski N
,
Furukawa H
.
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Since it was discovered that the anti-hypertensive agent ifenprodil has neuroprotective activity through its effects on NMDA (N-methyl-D-aspartate) receptors, a determined effort has been made to understand the mechanism of action and to develop improved therapeutic compounds on the basis of this knowledge. Neurotransmission mediated by NMDA receptors is essential for basic brain development and function. These receptors form heteromeric ion channels and become activated after concurrent binding of glycine and glutamate to the GluN1 and GluN2 subunits, respectively. A functional hallmark of NMDA receptors is that their ion-channel activity is allosterically regulated by binding of small compounds to the amino-terminal domain (ATD) in a subtype-specific manner. Ifenprodil and related phenylethanolamine compounds, which specifically inhibit GluN1 and GluN2B NMDA receptors, have been intensely studied for their potential use in the treatment of various neurological disorders and diseases, including depression, Alzheimer's disease and Parkinson's disease. Despite considerable enthusiasm, mechanisms underlying the recognition of phenylethanolamines and ATD-mediated allosteric inhibition remain limited owing to a lack of structural information. Here we report that the GluN1 and GluN2B ATDs form a heterodimer and that phenylethanolamine binds at the interface between GluN1 and GluN2B, rather than within the GluN2B cleft. The crystal structure of the heterodimer formed between the GluN1b ATD from Xenopus laevis and the GluN2B ATD from Rattus norvegicus shows a highly distinct pattern of subunit arrangement that is different from the arrangements observed in homodimeric non-NMDA receptors and reveals the molecular determinants for phenylethanolamine binding. Restriction of domain movement in the bi-lobed structure of the GluN2B ATD, by engineering of an inter-subunit disulphide bond, markedly decreases sensitivity to ifenprodil, indicating that conformational freedom in the GluN2B ATD is essential for ifenprodil-mediated allosteric inhibition of NMDA receptors. These findings pave the way for improving the design of subtype-specific compounds with therapeutic value for neurological disorders and diseases.
Figure 2. Structure of GluN1b/GluN2B ATD heterodimer in complex with ifenprodil at 2.6 Ã resolutiona, View from side of amino termini. GluN1b and GluN2B ATDs have bi-lobed architecture composed of R1 (magenta and cyan) and R2 (light pink and yellow) domains. Ifenprodil (gray sphere) sits at the heterodimer interface. N-glycosylation hains are shown in white. The cartoon shows an approximate orientation of the GluN1b and GluN2B ATDs with black sticks below R2 indicating the C-terminal ends where ligand-binding domains (LBDs) begin. b, Surface presentation of the GluN1b/GluN2B ATD heterodimer (upper panel) and each subunit (lower panel) showing residues at the subunit interface (dark blue). Note that ifenprodil (gray sphere) is occluded in the subunit interface. The heterodimer buries 1,191 Ã 2 of solvent accessible surface area per subunit with GluN1b R1-GluN2B R1 and GluN1b R1-GluN2B R2 interfaces contributing 62% and 38%, respectively.
Figure 3. Phenylethanolamine binding sitea-b, Bindings of ifenprodil (a) and Ro 25-6981 (b) take place at the GluN1b-GluN2B subunit interface. Mesh represents Fo-Fc omit electron density map contoured at 3Ï. Residues with asterisks in panel a are the ones previously shown to affect ifenprodil sensitivities. Adjacent to the binding pocket is an empty space surrounded by hydrophobic residues including GluN1b Ala75, GluN2B Ile82 and Phe114 (arrows). c, Comparison of binding patterns of ifenprodil (gray) and Ro 25-6981 (lime) in stereoview. The Ro 25-6981-bound structure is coloured as in panel b whereas the ifenprodil-bound structure is coloured white. d, New residues found to interact with phenylethanolamines in this study are mutated and analyzed for sensitivity to ifenprodil. Mutation of the residues surrounding the binding site caused changes in IC50 as well as extent of inhibition.
Figure 4. Engineering of disulphide bonds at the subunit interface alters ifenprodil sensitivitya, Location of mutated residues at the R1-R1 and R1-R2 interfaces in GluN1b/GluN2B ATDs (sphere) and the ifenprodil binding pocket (asterisk). b, Observation of disulphide bonds by anti-GluN1 and anti-GluN2B blots in reducing (DTT) and non-reducing (âDTT) conditions. c, Macroscopic current recording of the wild-type and mutant receptors in the presence (red) and absence (black) of DTT (2 mM). d, Effect of disulphide bonds on ifenprodil sensitivity in the presence (red) and absence (black) of DTT. e, Possible model of ifenprodil binding and movement of ATD for allosteric inhibition. Ifenprodil binds to the open GluN2B clamshell and induces domain closure thereby favouring resulting in allosteric inhibition. In the GluN1-4b (N70C)/GluN2B (T324C) receptors, the GluN2B ATD is locked in the closed conformation, thus, ifenprodil cannot access the binding site.
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