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. 2017 Apr 5;7:44578.
doi: 10.1038/srep44578.

Computationally Discovered Potentiating Role of Glycans on NMDA Receptors

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Free PMC article

Computationally Discovered Potentiating Role of Glycans on NMDA Receptors

Anton V Sinitskiy et al. Sci Rep. .
Free PMC article

Abstract

N-methyl-D-aspartate receptors (NMDARs) are glycoproteins in the brain central to learning and memory. The effects of glycosylation on the structure and dynamics of NMDARs are largely unknown. In this work, we use extensive molecular dynamics simulations of GluN1 and GluN2B ligand binding domains (LBDs) of NMDARs to investigate these effects. Our simulations predict that intra-domain interactions involving the glycan attached to residue GluN1-N440 stabilize closed-clamshell conformations of the GluN1 LBD. The glycan on GluN2B-N688 shows a similar, though weaker, effect. Based on these results, and assuming the transferability of the results of LBD simulations to the full receptor, we predict that glycans at GluN1-N440 might play a potentiator role in NMDARs. To validate this prediction, we perform electrophysiological analysis of full-length NMDARs with a glycosylation-preventing GluN1-N440Q mutation, and demonstrate an increase in the glycine EC50 value. Overall, our results suggest an intramolecular potentiating role of glycans on NMDA receptors.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
(a) Ligand binding domains (LBD) of GluN1 (blue) and GluN2B (green) subunits are parts of an NMDA receptor (gray, blue and green; protein part only shown). Each NMDAR contains two GluN1 and two GluN2B LBDs. The overall architecture of NMDARs is annotated on the right: the amino terminal domain (ATD) and the LBD are extracellular parts, the transmembrane domain (TMD) is immersed into the lipid bilayer, and the carboxyl terminal domain (CTD, not resolved in X-ray structures) is a cytosolic part. (b) For computational feasibility, this work focuses on the independent GluN1 LBD and GluN2B LBD. (c) Three Man5 glycans (red) were attached to GluN1 LBD and three Man5 glycans to GluN2B LBD to match the glycosylation pattern of NMDARs in the brain.
Figure 2
Figure 2
(a,b) The physiologically most important conformational changes in the GluN1 (a) and GluN2B (b) LBDs are believed to be clamshell-like opening/closing motions, which can be quantified, for example, by changes in the distance d between Cα atoms in residues 507 and 701 in GluN1 or residues 503 and 701 in GluN2B (gray, van-der-Waals spheres). Protein is shown in cartoon representation; glycans, van-der-Waals spheres. (c) Glycosylation stabilizes closed conformations of the GluN1 LBD, though open conformations are still populated. (P.d.f.: probability distribution function). (d) A cartoon representation of the clamshell-like opening/closing motion in LBDs, with open conformations corresponding to larger values of d in panels (c,e). (e) Glycosylation of the GluN2B LBD stabilizes closed-clamshell conformations as well, though this effect is less pronounced as in GluN1 LBD.
Figure 3
Figure 3
(a) Man5 glycan at residue GluN1-N440 (van-der-Waals spheres, right) and amino acid residues Glu712, Glu716, Gln719 and Asp723 from the other lobe of GluN1 LBD (green) transiently noncovalently interact, which explains why closed conformations of the GluN1 LBD are more stable in the glycosylated state. (b) In open conformations of the GluN1 LBD, the Man5 glycan and the other lobe of the glycoprotein do not interact. (c) Representative structures for the transient interactions between Man5 glycan (CPK representation) and residues 712, 716, 719, 723 (van-der-Waals spheres) illustrate that no single stable structure exists under physiological conditions.
Figure 4
Figure 4. Glycan attached to GluN1-N440 interacts with the opposite lobe of the protein only in closed-clamshell conformations of GluN1 LBD.
This heatmap shows a two-dimensional distribution of geometries of glycosylated GluN1 LBD in all 536,651 frames of 262 MD trajectories in terms of two variables: d, measuring whether a conformation is clamshell open/closed, and dg-ol, the shortest distance between heavy atoms in the glycan attached to N440 and heavy atoms in the other lobe of the protein (residues 710 to 723). (Note the log scales on the y axis and the colorbar). The empty field in the region of the diagram with d > 5.2 nm and dg-ol < 0.5 nm implies that no open-clamshell conformations with the glycan interacting with the opposite lobe of the protein have been reached.
Figure 5
Figure 5. The mutation GluN1-N440Q in the GluN1/GluN2A NMDA receptor results in a small but detectible rightward shift in the glycine EC50.
(A,B) The wild-type and N-to-Q mutant channels were expressed in oocytes and the dose response was measured using two-electrode voltage clamp recordings. (C) Averaged data from n = 12 recordings of each were plotted and fit with a Hill function to reveal a 50% increase in the glycine EC50 in the presence of the N440Q mutation, which makes glycosylation at this residue impossible.

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