Mechanistic insights into xenon inhibition of NMDA receptors from MD simulations

Abstract

Inhibition of N-methyl-D-aspartate (NMDA) receptors has been viewed as a primary cause of xenon anesthesia, yet the mechanism is unclear. Here, we investigated interactions between xenon and the ligand-binding domain (LBD) of a NMDA receptor and examined xenon-induced structural and dynamical changes that are relevant to functional changes of the NMDA receptor. Several comparative molecular dynamics simulations were performed on two X-ray structures representing the open- and closed-cleft LBD of the NMDA receptor. We identified plausible xenon action sites in the LBD, including those nearby agonist sites, in the hinge region, and at the interface between two subunits. The xenon-binding energy varies from -5.3 to -0.7 kcal/mol. Xenon's effect on the NMDA receptor is conformation-dependent and is produced through both competitive and noncompetitive mechanisms. Xenon can promote cleft opening in the absence of agonists and consequently stabilizes the closed channel. Xenon can also bind at the interface of two subunits, alter the intersubunit interaction, and lead to a reduction of the distance between two GT linkers. This reduction corresponds to a rearrangement of the channel toward a direction of pore size decreasing, implying a closed or desensitized channel. In addition to these noncompetitive actions, xenon was found to weaken the glutamate binding, which could lead to low agonist efficacy and appear as competitive inhibition.

Figure 1

Top (A) and side (B) views of xenon trajectories in the closed-cleft ligand-binding domain of the NMDA receptor (PDB code: 2A5T) over a 20-ns simulation. The black vector represents a pseudo two fold symmetric axis between the two subunits, NR1 (white) and NR2 (gray). The agonist glycine (LG) and glutamate (LE) are represented in stick. Initial xenon locations in the simulation are marked with spheres. Seven xenon atoms: Xe-1 in brown, flanked by helix-F and helix-G of NR1; Xe-2 in pink, flanked by helix-F and helix-G of NR2; Xe-3 in blue, flanked by helix-I and helix-K of NR1. Xe-4 (green) and Xe-5 (cyan) are next to the glutamate and glycine agonists, respectively. Xe-6 (orange) and Xe-7 (red) are located at the interface of NR1 and NR2. The xenon trajectories are shown in solid lines with the time step of 10 ps. For clarity, on the right, the xenon atoms and two agonists are labeled. The trajectories for Xe-2, Xe-3, and Xe-7 are not shown because they moved out of the protein during the simulation.

Figure 2

The van der Waals interaction energies between Xe-4_closed and Xe-5_closed and their neighboring residues within 7 Å in the closed-cleft conformation of the NMDA receptor during the 20-ns simulation. The averaged van der Waals interaction energy computed for the last 5 ns is −7.05 (±0.68) kcal/mol for Xe-4_closed and −5.28 (±1.08) kcal/mol for Xe-5_closed, respectively.

Figure 3

Snapshots from the FEP calculation when the interaction of Xe-4_closed (green sphere) with its surrounding was completely annihilated (A); partially annihilated (B); and fully included (C). The more the xenon interactions are included, the less hydrogen bonding (black dash line) around the glutamate (colored by atom type in transparent surface). All the residues within 5 Å away from glutamate are shown in stick. In the FEP calculation, a xenon atom is annihilated gradually, which leads to the xenon atom migrating out its original site (A) due to the disappearing interaction with other atoms. To evaluate the change of the original site in this process, we picked the relative stable glutamate, ~ 3 Å to Xe-4_closed in (C), as the reference of this site.

Figure 4

(A) The structure of a ligand-binding domain (green) showing the definition of the domain closure. The blue sphere represents the hinge, the red and black vectors point to the center of mass of Domain 1 and Domain 2, respectively. The angle between the two vectors defines the domain closure. To prevent the bias from flexible loops, only the α helices of Domain 1 were included in the calculation of the center of mass. (B) Comparison of the domain closure in the open-cleft (1PBQ-NR1A & 1PBQ-NR1B) and closed-cleft (2A5T-NR1 & 2A5T-NR2) conformations in the absence (blue) or presence (red) of xenon at the first 0.5-ns (light color) and last 5-ns (dark color) MD simulations. Mean standard deviations were calculated using 50 or 500 frames from the first 0.5-ns or last 5-ns simulation, respectively.

Figure 5

Difference of backbone flexibility between the xenon and control systems for the ligand-binding domain of the NMDA receptor in the closed-cleft conformation (cartoon) is highlighted using a color gradient: from the most increased flexibility (red) to the most decreased flexibility (blue). (A) Top and (B) site views of the RMSF changes in the presence and absence of xenon over the last 5-ns simulations. (C) Top and (D) site views of the changes based on GNM analysis in the mean square fluctuation of the three lowest motions in the systems with and without xenon atoms. The glycine (LG) and glutamate (LE) agonists are presented in solid surface. Domain-1 is colored silver in both subunits. Domain-2 is colored in yellow in NR1 and light blue in NR2. The disulphide bonds in Loop 1 are highlighted in transparent surface and colored by atom type. Xenon atoms were not included in the GNM network, as explained in the Supporting Information by Fig. S6.

Figure 6

(A) The locations of the GT linkers in the initial (transparent in white) and final (solid) structures of NR1 (yellow) and NR2 (blue) in the simulation. The two agonists are presented in surface and colored by atom types. (B) The distance between the GT linkers of NR1 and NR2 subunits over 20-ns simulations in the absence (black) or presence (red) of xenon atoms. A GT linker is where the ligand-binding domain is connected with the TM domains of the NMDA receptor (see Fig. S1).

Figure 7

A diagram showing how xenon affects the domain closure of the ligand-binding domain (LBD) of the NMDA receptor. Multiple conformations exist with various degrees of domain closure, such as C, O1, O2 and O3. Agonist binding initiates the S1S2 cleft closing (C) and the channel opening. Xenon may weaken the agonist binding, but is unable to replace the agonist and open the cleft. Xenon can also inhibit the channel in a non-competitive manner by altering the interaction between NR1 and NR2 (not shown here). For LBD without agonists, xenon may promote the cleft opening from O1 to O2 or from O2 to O3. When the S1S2 cleft is widely opened (O3) by xenon, the separation between two domains is too large to allow agonist binding at the hinge region of the S1S2 cleft. This is another format of non-competitive xenon inhibition. The competitive inhibition can occur between two intermediate states O1 and O2: xenon promotes the transition from O1 to O2, while agonists work in the opposite direction.