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    • The LBD experiences a large scale conformational

    2022-01-19

    The LBD experiences a large-scale conformational transition between open and closed states upon agonist binding within the cleft separating the two lobes of the domain. The upper lobe (Lobe 1, also called D1) is connected to the ATD, and the lower lobe (Lobe 2, also called D2) is connected to the TMD. The first high-resolution structure of a tetrameric “full-length” (ATD-LBD-TMD), or intact, iGluR showed how the dimer-of-dimers arrangement of the LBDs could allow LBD closure to drive opening of the channel pore [6] (Fig. 1). Prior and subsequent to this structure, however, structures of individual iGluR LBDs and ATDs have helped characterize the conformational changes that take place within these domains upon the binding of various ligands, including agonists, antagonists, and allosteric modulators [7]. LBD dimer structures have also helped characterize the molecular rearrangements responsible for receptor desensitization [8]. The structures of isolated domains have represented the four iGluR families in vertebrates: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic TCS 3035 (AMPA), kainate (GluK), N-methyl-d-aspartate (NMDA), and δ-receptors. These receptors resemble each other in overall architecture but have been found to exhibit marked functional differences, e.g., in activation and desensitization kinetics and in maximum open probabilities [9]. NMDA receptors are unique in requiring both glutamate and glycine (or d-serine) for activation. The wealth of information provided by high-resolution structures of these isolated domains in apo and holo states have presented opportunities for molecular simulation studies, most of which have concerned the LBD. Early conventional molecular dynamics (MD) simulations of the GluA2 (named GluR2 at the time) AMPA receptor LBD provided insights into functionally relevant conformational fluctuations in the LBD for apo and ligand-bound states [10,11]. These simulations were, of course, limited by the computational power available in the early 2000s. An alternative approach to carrying out simulation studies involves the use of enhanced sampling methods [12,13]. These methods allow one to examine specific molecular events, such as conformational transitions, that occur on timescales difficult to access by conventional MD simulations. Boltzmann distributions of conformational ensembles consistent with what would be obtained by conventional simulations is obtained, which permits free energies associated with the molecular events to be computed. These free energies can then be directly compared with appropriate experimental measurements.
    AMPA receptor LBD The first computations of the free energy landscapes, or potentials of mean force (PMFs), that govern large-scale conformational changes in an iGluR were performed for the GluA2 LBD [14]. The PMF provides a reduced description of conformational changes in a system along a set of chosen coordinates [15] (Fig. 2A). The difference in free energy between conformational states reflects the relative probability of the system populating those states. The simulation strategy used in this study was umbrella sampling [16], which features prominently in this review. Briefly, umbrella sampling involves running multiple independent MD simulations where each simulation is under the influence of an applied energetic bias that restrains the system to a specific region, or window, of conformational space. The sampling data from all windows are subsequently unbiased and combined using techniques such as the weighted histogram analysis method (WHAM) [17] or multistate Bennett acceptance ratio (MBAR) [18] to generate the PMF (Fig. 2B). The PMFs suggest that the LBD, in isolation, exists not in rigid conformations when in the apo form or when bound to an agonist or antagonist, but populates a range of conformations to varying degrees, with the apo LBD easily adopting more open cleft conformations than observed in crystal structures [19]. The range of conformations predicted to be sampled by the LBD is consistent with experimentally measured SAXS data [20]. These conformations were later found to be consistent with single-molecule FRET (smFRET) measurements [21]. Analysis of the simulation data also suggested that a dense cluster of water molecules within the apo binding cleft may stabilize the open conformation. Upon glutamate binding, 9–12 kcal/mol of free energy is predicted to become available for driving the conformational changes in the LBD associated with receptor activation.