Ionotropic glutamate receptors (iGluRs) are tetrameric protein complexes that transduce chemical signals carried by neurotransmitter molecules into electrical impulses propagated in the postsynaptic neuron. Each protein subunit includes an amino-terminal domain (ATD) and a cytoplasmic carboxy-terminal domain (CTD) involved in receptor assembly, trafficking and regulation, a transmembrane domain (TMD) forming the membrane-spanning ion channel, and a ligand-binding domain (LBD) which is key to channel gating1
. The binding of agonist molecules to the LBDs drives the opening of the transmembrane pore, allowing cations to flow across the cell membrane to trigger the generation of a nerve impulse. Full agonists such as glutamate display the highest observed levels of efficacy at the receptor, while antagonists block receptor activation, and partial agonists produce sub-maximal response when applied at saturating concentrations.
The LBD is a flexible clamshell-shaped protein, which makes a conformational transition from an open to a closed state upon the binding of an agonist molecule into the cleft separating its two lobes. Four LBDs are tethered to the TMD via short linkers, and once the LBDs close down to encapsulate the ligand, the local conformational change is assumed to force the opening of the TM channel2
. A central issue is to understand how the binding of different ligands leads to, or inhibits, the activation of the receptor. One possible mechanism, inferred from crystal structures of LBD–ligand complexes, is that ligand efficacy is directly correlated with the amount of cleft closure induced by the bound ligand3
. There are discrepancies, however, which are not fully understood. For example, the LBD of NR1 and GluK5 subunits have been shown to close fully even when bound to partial agonists4,5
. The relative twist between the two lobes has also been suggested to be important6–8
. Such observations suggest that a purely “structural” explanation, based solely on cleft closure is not completely satisfactory.
Alternatively, a “dynamical” mechanistic perspective might be that full agonists succeed in tightly closing down the clamshell via strong LBD–ligand interactions, while bound partial agonists exert only weak cleft-closing forces and are thus unable to prevent transient fluctuations leading to partial re-openings of the LBD. The measured binding affinities of some antagonists to the isolated GluA2 LBD are stronger than the affinities of some agonists, which suggest that only a fraction of the total binding free energy is available to close the LBD and activate the receptor3,9
. A number of studies have shown that the efficacy of an agonist can also be modulated by the stability of the closed LBD–agonist complex10–13
A contrast can be drawn between the “structural” and “dynamical” views, where function is either explained by X-ray structures of the LBD in complex with different ligands or by the fluctuations and transient excursions of the LBD away from a static conformation, although both are necessarily oversimplified. Nevertheless, it is difficult to achieve a deeper understanding of the mechanism of activation of iGluR receptors without a detailed dissection of the different thermodynamic contributions associated with ligand-binding and LBD closure, which provides the link between structure and dynamics. Although central to understanding the activation mechanism of ligand-gated receptors, such thermodynamic information is difficult to access directly by experimental means, and remains essentially “hidden” from direct observations. In this paper, the free energy contributions governing the distinct sub-processes of ligand-docking and LBD closure for the GluA2 receptor from Rattus norvegicus are determined for nine different ligands using all-atom molecular dynamics (MD) simulations with explicit solvent molecules. The results are then used to carry out an analysis of LBD conformational distributions in the context of a full-length receptor, revealing key structural asymmetries that may impact activation.