Excitatory and inhibitory D-serine binding to the NMDA receptor
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Evaluation Summary:
Activation of NMDA receptors requires two co-agonists: Glutamate which binds to the GluN2 subunit and glycine/D-serine which binds to the GluN1 subunit. In the present manuscript, the authors address the interaction of D-serine, which is a less studied co-agonist than glycine, with the GluN1 and GluN2A subunits using molecular simulations as well as electrophysiology experiments. Surprisingly they find that D-serine interacts with the GluN2 subunit, further expanding our molecular understanding of NMDA receptor structure-function.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
N-methyl-D-aspartate receptors (NMDARs) uniquely require binding of two different neurotransmitter agonists for synaptic transmission. D-serine and glycine bind to one subunit, GluN1, while glutamate binds to the other, GluN2. These agonists bind to the receptor’s bi-lobed ligand-binding domains (LBDs), which close around the agonist during receptor activation. To better understand the unexplored mechanisms by which D-serine contributes to receptor activation, we performed multi-microsecond molecular dynamics simulations of the GluN1/GluN2A LBD dimer with free D-serine and glutamate agonists. Surprisingly, we observed D-serine binding to both GluN1 and GluN2A LBDs, suggesting that D-serine competes with glutamate for binding to GluN2A. This mechanism is confirmed by our electrophysiology experiments, which show that D-serine is indeed inhibitory at high concentrations. Although free energy calculations indicate that D-serine stabilizes the closed GluN2A LBD, its inhibitory behavior suggests that it either does not remain bound long enough or does not generate sufficient force for ion channel gating. We developed a workflow using pathway similarity analysis to identify groups of residues working together to promote binding. These conformation-dependent pathways were not significantly impacted by the presence of N-linked glycans, which act primarily by interacting with the LBD bottom lobe to stabilize the closed LBD.
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Evaluation Summary:
Activation of NMDA receptors requires two co-agonists: Glutamate which binds to the GluN2 subunit and glycine/D-serine which binds to the GluN1 subunit. In the present manuscript, the authors address the interaction of D-serine, which is a less studied co-agonist than glycine, with the GluN1 and GluN2A subunits using molecular simulations as well as electrophysiology experiments. Surprisingly they find that D-serine interacts with the GluN2 subunit, further expanding our molecular understanding of NMDA receptor structure-function.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
This is a study that is aimed at understanding the binding mechanism of D-serine to the two different binding lobes of the NMDA receptor. D-serine is a known agonist and binder of the GluN1 ligand-binding domain, but its interaction with the GluN2A is unknown. Using long time-scale conventional molecular dynamics simulations, the researchers observe that D-serine interacts and associates readily with both binding domains, often via protein surface pathways referred to as a guided-diffusion mechanism. As observed previously, free-energy calculations show that D-serine stabilizes the closure of both binding domains. Finally, analysis of the effect of glycans shows that these modifications play a role in further stabilizing the closed state of the ligand-binding domains.
Amongst this broad and careful analysis, …
Reviewer #1 (Public Review):
This is a study that is aimed at understanding the binding mechanism of D-serine to the two different binding lobes of the NMDA receptor. D-serine is a known agonist and binder of the GluN1 ligand-binding domain, but its interaction with the GluN2A is unknown. Using long time-scale conventional molecular dynamics simulations, the researchers observe that D-serine interacts and associates readily with both binding domains, often via protein surface pathways referred to as a guided-diffusion mechanism. As observed previously, free-energy calculations show that D-serine stabilizes the closure of both binding domains. Finally, analysis of the effect of glycans shows that these modifications play a role in further stabilizing the closed state of the ligand-binding domains.
Amongst this broad and careful analysis, the major finding from this work is that D-serine surprisingly associates with GluN2A, which has been known to bind glutamate to enable activation of the channel. Since the binding of D-serine to GluN2A had not been observed previously, they proposed that D-serine acts as an inhibitor for glutamate at high concentrations. This hypothesis was investigated and supported by electrophysiological experiments, yielding a novel result that presents new interpretations for the field. However, the guided-diffusion mechanism still remains hypothetical and is unclear as to whether this is in fact a driving force, or requirement, for the binding. Specifically, the following questions warrant further investigation:
1. Specific or non-specific association? It is possible that non-specific association events of ligands to the protein could be an intrinsic artifact of the MD simulations. To investigate this, it would be informative to compare the current results with a negative control simulation where the ligand was replaced with a similar amino acid or molecule that has been verified as a non-binder for NMDAR.
2. Dissociation events? Further clarification is required to understand whether any dissociation events are observed in these simulations to the non-specific sites or the final binding site. If dissociation is not observed, how does this impact the interpretation of the binding mechanisms that characterize only the association events?
3. Testing the hypothesis of guided diffusion. It is proposed that guided diffusion drives serine binding to its site. This would imply that the residues on this path are important, and if mutated, would decrease the association rate and the ability to compete with glutamate. Additional electrophysiological experiments or direct binding experiments would be useful in understanding the relevance of guided diffusion in the ligand-binding mechanism of NMDARs.
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