␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the primary mediators of fast excitatory synaptic transmission in the mammalian CNS. Structures of the extracellular ligandbinding domain suggest that the extent of cleft closure in the ligand-binding domain controls the extent of activation of the receptor. Here we have developed a fluorescence resonance energy transfer-based probe that allows us to study the extent of cleft closure in the isolated ligand-binding domain in solution. These investigations show that the wild-type protein exhibits a graded cleft closure that correlates to the extent of activation, which is in qualitative agreement with the crystal structures. However, the changes in extent of cleft closure between the apo and agonist-bound states are smaller than that observed in the crystal structures. We have also used this method to study the L650T mutant and show that in solution the ␣-amino-5-methyl-3-hydroxy-4-isoxazole propionate-bound form of this mutant exists primarily in a conformation that is more closed than predicted based on the activity, indicating that the degree of cleft closure alone cannot be used as a measure of extent of activation of the receptor, and there are possibly other mechanisms in addition to cleft closure that mediate the subtleties in extent of activation by a given agonist.fluorescence ͉ glutamate ͉ ion channel L igand-gated ion channels are allosteric proteins that convert chemical signals into electrical signals by forming transmembrane ion channels upon ligand binding to an extracellular domain. Ionotropic glutamate receptors, a member of the ligand-gated ion channels, serve as an excellent paradigm for studying allostery in this family of proteins (1-6). The crystal structures of the isolated ligand-binding domain of the GluR2 subunit of the ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor protein show a graded cleft closure with a direct correlation between the extent of cleft closure induced by a ligand and its extent of activation (5, 6), which is consistent with a multiple state-induced fit model. However, there is one exception to this correlation, the AMPA-bound form of the L650T mutation of the receptor crystallizes in two forms: one structure where the cleft is closed 11°and a second structure in which the cleft is closed 22°relative to the open apo form of the protein (7). These structures lie on either side of the cleft closure versus activation correlation observed for the wild-type protein, thus raising the question as to whether some ligands or modifications at the AMPA receptors can change the mechanism of activation from the multistate model to that defined by equilibrium of two distinct states.Here we have developed a fluorescence resonance energy transfer (FRET)-based assay that allows us to investigate the conformational changes in the ligand-binding domain in solution, thus allowing us to probe the conformational changes in this domain without the crystallographic constraints. In addition, we have u...

Glutamate receptors are the predominant mediators of excitatory synaptic signals in the central nervous system and are important in learning and memory as well as in diverse neuropathologies including epilepsy and ischemia. Their primary function is to receive the chemical signal glutamate (1), which binds to an extracellular domain in the receptor, and convert it into an electrical signal through the formation of cation-permeable transmembrane channels. Recently described end-state apo and ligated structures of the ligand-binding domain of a rat glutamate receptor provide a first view of specific molecular interactions between the ligand and the receptor that are central to the allosteric regulation of function in this protein. Yet there is little information on the mechanism and the structures of intermediates (if any) formed during the ligand-binding process. Here we have used time-resolved vibrational spectroscopy to show that the process involves a sequence of interleaved ligand and protein changes that starts with the docking of glutamate at the alpha-carboxylate moiety and ends with the establishment of the interactions between the gamma-carboxylate of glutamate and the protein.

Abstractα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the main excitatory neurotransmitter receptors in the mammalian central nervous system. Structures of the isolated ligand binding domain of this receptor have provided significant insight into the large scale conformational changes, which when propagated to the channel segments leads to receptor activation. However, in order to establish the role of specific molecular interactions in controlling such fine details as the magnitude of the functional response, we have used a multiscale approach, where changes at specific moieties of the agonists have been studied by vibrational spectroscopy, while large scale conformational changes have been studied using fluorescence resonance energy transfer (FRET) investigations. By exploiting the wide range of activations by the agonists, glutamate, kainate, and AMPA, for the wild type,Y450F, and L650T mutants and by using the multiscale investigation, we show that the strength of the interactions at the α-amine group of the agonist with the protein in all but one case tracks the extent of activation. Since the α-amine group forms bridging interactions at the cusp of the ligand binding cleft this appears to be a critical interaction through which the agonist controls the extent of activation of the receptor.α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, a member of the ionotropic glutamate receptor family, are the main mediators of fast excitatory synaptic transmission in the mammalian central nervous system. Signal transmission is initiated by glutamate binding to an extracellular ligand binding domain, which leads to the formation of cation specific transmembrane channels (1-6). Large scale expression of the isolated extracellular ligand binding domain (S1S2) has led the way for detailed structural studies of this domain. X-ray (7-10), nuclear magnetic resonance (NMR) (4,(11)(12)(13), and vibrational spectroscopic (14-16) investigations using this soluble protein have provided significant insight into the relationship between structure and function in this subtype.The structures of S1S2 show a graded cleft closure conformational change upon binding agonists with varying efficacy in the bilobed ligand binding domain (9,10). The extent of cleft closure induced by a given agonist in most cases exhibits a direct correlation to the extent of activation of the receptor, suggesting that this is one of the possible modes of coupling between the ligand binding domain and opening of the ion channel. Vibrational spectroscopic investigations using the S1S2 protein provide a more detailed view of the specific interactions between the agonist and the extracellular ligand binding domain and their role in the functioning of the receptor. The frequency shifts in the asymmetric carboxylate vibrational mode, which is sensitive to the strength of the non-covalent interactions at this moiety, indicate that partial *Address correspondence to: Vasanthi Jayaraman, Department of Integrative Biolo...

A novel truncated form (residues 1–214, with a randomized C-terminal tail) of the ligand-binding extracellular domain (ECD) of the human α1 glycine receptor (GlyR), with amino acids from the corresponding sequence of an acetylcholine binding protein (AChBP) substituted for two relatively hydrophobic membrane-proximal loops, was overexpressed using a baculovirus expression system. The mutant GlyR ECD, named GlyBP, was present in both soluble and membrane-associated fractions after cell lysis, though only the latter appeared to be in a native-like conformation capable of binding strychnine, a GlyR specific antagonist. The membrane-associated GlyBP was solubilized and detergent/lipid/protein micelles were affinity purified. After detergent removal, GlyBP may be isolated in either aqueous or vesicular form. Binding assays and spectroscopic studies using circular dichroism and FRET are consistent with both forms adopting equivalent native-like conformations. Thus GlyBP may be isolated as a soluble or membrane-associated assembly that serves as a structural and functional homolog of the ECD of GlyR.