Population Shift Mechanism for Partial Agonism of AMPA Receptor

Abstract

a-amino-3-hydroxy-5-methyl-4-isoaxazolepropionic acid (AMPA) ionotropic glutamate receptors mediate fast excitatory neurotransmission in the central nervous system, and their dysfunction is associated with neurological diseases. Glutamate binding to ligand-binding domains (LBDs) of AMPA receptors induces channel opening in the transmembrane domains of the receptors. The T686A mutation reduces glutamate efficacy so that the glutamate behaves as a partial agonist. The crystal structures of wild-type and mutant LBDs are very similar and cannot account for the observed behavior. To elucidate the molecular mechanism inducing partial agonism of the T686A mutant, we computed the free-energy landscapes governing GluA2 LBD closure using replica-exchange umbrella sampling simulations. A semiclosed state, not observed in crystal structures, appears in the mutant during simulation. In this state, the LBD cleft opens slightly because of breaking of interlobe hydrogen bonds, reducing the efficiency of channel opening. The energy difference between the LBD closed and semiclosed states is small, and transitions between the two states would occur by thermal fluctuations. Evidently, glutamate binding to the T686A mutant induces a population shift from a closed to a semiclosed state, explaining the partial agonism in the AMPA receptor.FIGURE 5 Free-energy landscapes of the GluA2 LBD along ðx 3 ;x 4 Þ. (A) Definition of a reaction coordinate related to hydrogen-bonding interactions between the D1 and D2 lobes. x 4 describes the distance between the COMs of Y450-G451 in the D1 lobe and D651-S652 in the D2 lobe. x 1 is also shown for comparison with x 4 . The free-energy landscapes along x 3 and x 4 for flipped (B) and unflipped (C) conformations are shown. White circles represent the corresponding crystal structures. A white triangle represents the average structure of the semiclosed state. The red dashed arrow on the landscape for T686A-Glu represents the direction along the reduced coordinate x 34 ¼ 0:26x 3 À 0:74x 4 . To see this figure in color, go online.

Structural Mechanisms Underlying Activity Changes in an AMPA-type Glutamate Receptor Induced by Substitutions in Its Ligand-Binding Domain

“…Because all the WT and T686S/A LBDs had a population of >88% in state 4 (Figure 5A and Table 4), the observed chemical shift differences between the LBD variants should predominantly reflect the structural differences in state 4. Our MD simulation study revealed that the cleft-locked T686A LBD forms conformations with longer lobe 1-lobe 2 distances than that in the crystal structure (Oshima et al, 2019). Combined with the fact that the lobe 1-lobe 2 interfacial residues showed ms-ms timescale dynamics in the relaxation dispersion experiment, LBDs in state 4 would exchange between two conformational states: cleft-fully-closed and -locked state (state 4A in Figure S4A) and cleft-partially-open state (state 4B in Figure S4A).…”

“…This cleft-semi-closed state cannot be distinguished from the cleftfully-closed state by the HD exchange experiments, because S654 and T655 form hydrogen bonds with the bound ligand in both states. The cleft of the semi-closed conformation was closed to the same degree as was the LBD complexed with the partial agonist (S)-4-AHCP, which in turn is 3 further closed than the LBD-kainate complex (Nielsen et al, 2005;Oshima et al, 2019;Pohlsgaard et al, 2011). The inter-LBD distance of the cleft-semi-closed LBD dimer was $5 Å shorter than that of the cleft-fully-closed state.…”

“…cleft-semi-closed LBD conformation was observed for the T686A LBD in state 3 (Figure S4A) (Oshima et al, 2019). This cleft-semi-closed state cannot be distinguished from the cleftfully-closed state by the HD exchange experiments, because S654 and T655 form hydrogen bonds with the bound ligand in both states.…”

“…The conformational change from the cleft-fully-closed LBD to the cleftpartially-open LBD would also serve to weaken the strain in the linker, as the inter-LBD distance of the cleft-partially-open LBDs in state 4B ACT was shown to be shorter than that of the cleft-fully-closed LBDs in state 4A ACT by our MD simulation The HD exchange rate was over the detection limit. (Oshima et al, 2019). Consequently, T686S/A-induced population shifts of LBD would contribute to slow desensitization by reducing the population of state 4A ACT from which AMPAR desensitizes.…”

Computational Structural Biology: Successes, Future Directions, and Challenges

“…Further examples that combine experiments with computations include uncovering the isoform-specific signatures in Ras interactions [93], redefining the protein kinase conformational space with machine learning [94], developing covalent inhibitors of epidermal growth factor receptor (EGFR) [95], proposing c-Jun N-terminal kinase (JNK) signaling as a therapeutic target for Alzheimer’s disease [96,97], describing bacterial Ras/Rap1 site-specific endopeptidase cleavage of Ras disrupting Ras/extracellular signal-regulated kinase (ERK) signaling through an atypical mechanism [98], and finally, the remarkable observation of the biased antagonism of the CXC chemokine receptor type 4 (CXCR4) [99]. Additional examples, not detailed here are described in other works [34,100,101,102,103,104,105,106,107,108,109,110,111].…”