Activation and desensitization of ionotropic glutamate receptors by selectively triggering pre-existing motions

Abstract

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that are key players in synaptic transmission and plasticity. They are composed of four subunits, each containing four functional domains, the quaternary packing and collective structural dynamics of which are important determinants of their molecular mechanism of function. With the explosion of structural studies on different members of the family, including the structures of activated open channels, the mechanisms of action of these central signaling machines are now being elucidated. We review the current state of computational studies on two major members of the family, AMPA and NMDA receptors, with focus on molecular simulations and elastic network model analyses that have provided insights into the coupled movements of extracellular and transmembrane domains. We describe the newly emerging mechanisms of activation, allosteric signaling and desensitization, as mainly a selective triggering of pre-existing soft motions, as deduced from computational models and analyses that leverage structural data on intact AMPA and NMDA receptors in different states.

Keywords: AMPA and NMDA receptors; Activation mechanism; Allosteric interactions; Desensitization; Elastic network models; Ionotropic glutamate receptors; Molecular dynamics; Simulations.

Graphical abstract

Fig. 1

iGluRs are made of periplasmic binding protein (PBP) domains that close and undergo dimeric rearrangements. (A) The ligand-binding domain (LBD) is a clamshell domain that closes around the agonist glutamate. Shown are single subunits from LBD crystal structures in the ligand-free (apo) and glutamate-bound states together with their protein databank (PDB) codes. (B) LBDs form dimers with the two clamshells back to back. Closure of the two clefts pulls apart either the lower lobes to pull open the channel (activation) or the upper lobes to decouple from channel opening and remain closed (desensitization). (C) The NTD could also be produced as a separate module whose structure could be solved by X-ray crystallography. Two structures are shown representing the two main conformations and subfamilies. (D) A structure of the whole receptor is shown that is based on the resting state structure from cryo-EM (PDB 4UQJ), along with a schematic representation (on the right). Some linkers and the C-termini were modelled in with MODELLER.

Fig. 2

Two main methods for computational analysis of protein dynamics are illustrated. (A) All-atom molecular dynamics (MD) simulations include all atoms from the protein as well as surrounding water, salt and membrane. Shown is another model of a whole GluA2 AMPAR homotetramer including the C-terminal tails, generated using MODELLER and the CHARMM-GUI (785,175 atoms, of which 54,176 belong to the protein). The inset shows a zoomed view of three amino acids that are interacting and the myriad interactions between all their atoms. Covalent bonds are indicated by thick black dashed lines, Coulomb electrostatic interactions are yellow and van der Waals contacts are grey. (B) An elastic network model (ENM) representation is shown for the same AMPAR model with the C-tails removed. It has a node for each residue (a total of 3168 nodes). Uniform springs connect residues within 15 Å (thick black lines). The three residues shown in panel A are now represented by three nodes with springs describing their interactions, which oscillate around the starting distances shown.

Fig. 3

Global dynamics of iGluRs related to allosteric inhibition and desensitization. (A) A conformation generated by traversing ANM mode 4 of the original AMPAR crystal structure (PDB 3KG2) in one direction shows a compact packing of the NTD and LBD, which is highlighted in red and blue for the two pairs of NTD dimers and LBD dimers. The lower diagram displays the relative positions of the NTD dimers, which reveal a compact O shape when viewed from the extracellular region. (B) A similar compaction of the NTD and LBD is observed in the allosterically inhibited NMDAR crystal structure (PDB 4PE5) and a similarly compacted NTD arrangement is seen in the top view. (C-D) Traversing this ANM mode back in the other direction, we pass through the starting structure (C) where the NTD forms the classical N shape, and then reach an alternative conformation with the NTD dimers lifted away from the LBD and tilted away from each other and from the central symmetry axis similar to low resolution desensitized structures (D). See Movie 1 for more details, including cross-sectional views of the NTD, LBD and TMD layers.

Fig. 4

Global dynamics of iGluRs related to activation. (A-B) Comparison of the original AMPAR structure (PDB 3KG2) (A) and ANM intermediate along mode 6 (B) shows an iris-like rotation and opening of the TMD and NTD inter-dimer pivoting coupled to complex LBD dynamics, involving a rolling of the LBD dimers towards and away from each other together with a sliding rotation past each other. Top views of the LBD and TMD are displayed in each panel, along with color-coded arrows to more clearly show the collective motions of the four subunits and three layers (NTD, LBD and TMD) in mode 6. The latter can be viewed more clearly in Movie 2. (C) The TARP-associated open channel state (PBD 5WEO) reveals similar motions of the LBD and TMD but minimal NTD dynamics as a result of the modified NTD-LBD linker present in the crystallography construct used. The TARPs are shown in surface representation to highlight their optimized positioning for modulation of LBD and TMD dynamics. Black surfaces on the TARPs in the top view of the TMD layer indicate contacts with the LBD.