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. 2017 Nov 22;121(46):10436-10442.
doi: 10.1021/acs.jpcb.7b06862. Epub 2017 Nov 9.

Energetics of Glutamate Binding to an Ionotropic Glutamate Receptor

Alvin Yu  1   2 , Albert Y Lau  1   2
Affiliations

Energetics of Glutamate Binding to an Ionotropic Glutamate Receptor

Alvin Yu et al. J Phys Chem B. .

Abstract

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that are responsible for the majority of excitatory transmission at the synaptic cleft. Mechanically speaking, agonist binding to the ligand binding domain (LBD) activates the receptor by triggering a conformational change that is transmitted to the transmembrane region, opening the ion channel pore. We use fully atomistic molecular dynamics simulations to investigate the binding process in the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, an iGluR subtype. The string method with swarms of trajectories was applied to calculate the possible pathways glutamate traverses during ligand binding. Residues peripheral to the binding cleft are found to metastably bind the ligand prior to ligand entry into the binding pocket. Umbrella sampling simulations were performed to compute the free energy barriers along the binding pathways. The calculated free energy profiles demonstrate that metastable interactions contribute substantially to the energetics of ligand binding and form local minima in the overall free energy landscape. Protein-ligand interactions at sites outside of the orthosteric agonist-binding site may serve to lower the transition barriers of the binding process.

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Figures

Figure 1
Figure 1
Opening and closing of the GluA2 LBD is described by the two-dimensional order parameter (ξ1, ξ2). ξ1 is the center of mass distance between residues 479–481 on lobe 1 and residues 654–655 on lobe 2 (green). ξ2 is the center of mass distance between residues 401–403 on lobe 1 and residues 686–687 on lobe 2 (cyan). Together, they indicate the degree of cleft closure in the open-to-closed transition of the LBD as in Lau et al.,
Figure 2
Figure 2
Locally optimized binding intermediates in pathway 1. (A) The initial conformation used in the string method for pathway 1 contains an open LBD (ξ1, ξ2) = (14.1 Å, 13.9 Å) and a ligand separated by bulk solvent. (B) Close-up view of (A). (C) Glutamate contacts R684 on lobe 2. (D) The S652 hydroxyl sidechain hydrogen bonds with the amine of glutamate. (E) K449 interacts with the ligand's γ-carboxylate (F) Glutamate shifts into the binding pocket. (G) Glutamate forms three contacts across lobe 1 and lobe 2 of the LBD. (1) The α-carboxylate contacts the backbone amine of Y450. (2) The amine interacts with the S652 hydroxyl side chain. (3) K449 contacts the γ-carboxylate. (H) Glutamate is coordinated by the aromatic sidechain of Y450, aligning with the α-carboxylate proximal to R485. (I) The α-carboxylate contacts R485 on lobe 1 and E705 on lobe 2. (J-K) The LBD closes further around the ligand (ξ1, ξ2) = (9.5 Å, 8.3 Å) as the γ-carboxylate contacts the backbone amines of S654 and T655. (L) Expanded view of (K).
Figure 3
Figure 3
Locally optimized binding intermediates in pathway 2. (A) The initial conformation used in the string method for pathway 2 contains an open LBD (ξ1, ξ2) = (14.1 Å, 13.9 Å) and a ligand separated by bulk solvent. (B) Close-up view of (A). (C-D) Glutamate contacts R453 on the ξ1 side of lobe 1. (E) R661 contacts the ligand's γ-carboxylate (F) R485 flickers out of the binding pocket to contact the γ-carboxylate. (G-I) R485 relaxes toward the binding pocket while coordinating the ligand's γ-carboxylate. (J) The γ-carboxylate flips downward into the binding pocket, while the α-carboxylate flips upward to contact R485. The amine contacts E705 on lobe 2. (K) The LBD closes further around the ligand (ξ1, ξ2) = (9.5 Å, 8.3 Å) as the γ-carboxylate coordinates the backbone amines of S654 and T655. (L) Expanded view of (K).
Figure 4
Figure 4
The free energy profile along binding pathway 1. The indices refer to images, or conformations, along pathway 1, calculated by the string method. The free energy was calculated by Voronoi tessellating the phase space of the collective variables and performing umbrella sampling. At images (i = 90 – 129) the ligand is in bulk solvent, and at image 0, the ligand is fully bound within the LBD, in the crystallographic conformation. Protein-ligand interactions that change the energetics of ligand binding are labeled. The asterisk (*) indicates an image that contains a metastable intermediate consisting of the following interactions: ligand α-carboxylate to Y450 backbone amine, ligand amine to S652 hydroxyl side chain, and ligand γ-carboxylate to K449 sidechain.
Figure 5
Figure 5
The free energy profile along binding pathway 2. The indices refer to images, or conformations, along pathway 2, calculated by the string method. The free energy was calculated by Voronoi tessellating the phase space of the collective variables and performing umbrella sampling. At images (i = 102–131) the ligand is in bulk solvent, and at image 0, the ligand is fully bound within the LBD, in the crystallographic conformation. Protein-ligand interactions that change the energetics of ligand binding are labeled.
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