Structural Mobility of the Extracellular Ligand-Binding Core of an Ionotropic Glutamate Receptor. Analysis of NMR Relaxation Dynamics

In the present report, using vibrational spectroscopy we have probed the ligand-protein interactions for full agonists (glutamate and ␣-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA)) and a partial agonist (kainate) in the isolated ligand-binding domain of the GluR2 subunit of the glutamate receptor. These studies indicate differences in the strength of the interactions of the ␣-carboxylates for the various agonists, with kainate having the strongest interactions and glutamate having the weakest. Additionally, the interactions at the ␣-amine group of the agonists have also been probed by studying the environment of the non-disulfide-bonded Cys-425, which is in close proximity to the ␣-amine group. These investigations suggest that the interactions at the ␣-amine group are stronger for full agonists such as glutamate and AMPA as evidenced by the increase in the hydrogen bond strength at Cys-425. Partial agonists such as kainate do not change the environment of Cys-425 relative to the apo form, suggesting weak interactions at the ␣-amine group of kainate. In addition to probing the ligand environment, we have also investigated the changes in the secondary structure of the protein. Results clearly indicate that full agonists such as glutamate and AMPA induce similar secondary structural changes that are different from those of the partial agonist kainate; thus, a spectroscopic signature is provided for identifying the functional consequences of a specific ligand binding to this protein.Glutamate receptors are the predominant mediators of excitatory synaptic signals in the central nervous system (1-5). These receptors form cation-specific channels on binding glutamate and later enter a desensitized state in which the channel is closed, despite the presence of an agonist. Significant insights into the mechanism of agonist-induced activation and desensitization of this receptor have been obtained from the recent x-ray structures of the isolated, extracellular ligandbinding domain (S1S2) of the ␣-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA) 1 subtype of the glutamate receptors (6 -9). The x-ray structures of the S1S2 protein indicate a bilobed structure with the ligand-binding site located in the cleft between the lobes. The protein exists in an open structure in the apo state and undergoes varying degrees of cleft closure on binding various agonists. This cleft closure is believed to be a rigid body motion of one lobe relative to the other. Furthermore, the degree of lobe closure has been shown to be directly proportional to the extent of activation of these receptors, suggesting cleft closure in the S1S2 protein as the mechanism of activation of the receptor (7,8).The x-ray structures of the S1S2 protein in various ligated states have provided the first structural insights into the structure-function correlations in the glutamate receptor. These insights, however, are low temperature static images that are limited by the constraints of crystal-packing forces. For a more detailed investigation of spe...

Section: Resultssupporting

confidence: 88%

mentioning

confidence: 99%

Chemistry and Conformation of the Ligand-binding Domain of GluR2 Subtype of Glutamate Receptors

Cheng

Jayaraman

2004

“…Additionally, even in the closely related AMPA receptors there are several mutants and agonists that deviate from the cleft closure mechanism. Structural and spectroscopic investigations such as NMR (22,35,36,38) and single molecule FRET investigations (37) of these mutants do not support a shift to a two-state model but indicate the role of other mechanisms such as the dynamics of the protein and differences at the level of specific interactions such as hydrogen bonds that contribute to differences in activation by different ligands. Also, it should also be noted that although there is a correlation between the average cleft closure and activation for the GluN2 subunit, the dynamics and specific interactions at the level of side chains may also play a role in activation for this subunit.…”

Section: Resultsmentioning

confidence: 99%

Conformational Changes at the Agonist Binding Domain of the N-Methyl-d-Aspartic Acid Receptor

Rambhadran¹,

González²,

Jayaraman³

2011

The conformational changes in the agonist binding domain of the glycine-binding GluN1 and glutamate-binding GluN2A subunits of the N-methyl D-aspartic acid receptor upon binding agonists of varying efficacy have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances indicate a cleft closure conformational change at the GluN1 subunit upon binding agonists; however, no significant changes in the cleft closure are observed between partial and full agonists. This is consistent with the previously reported crystal structures for the isolated agonist binding domain of this receptor. Additionally, the LRET-based distances show that the agonist binding domain of the glutamate-binding GluN2A subunit exhibits a graded cleft closure with the extent of cleft closure being proportional to the extent of activation, indicating that the mechanism of activation in this subunit is similar to that of the glutamate binding ␣-amino-5-methyl-3-hydroxy-4-isoxazole propionate and kainate subtypes of the ionotropic glutamate receptors.Glutamate receptors are the main mediators of excitatory signal transmission in the mammalian central nervous system. Glutamate binding to an extracellular domain in the receptor triggers the formation of a cation-permeable transmembrane channel in the receptor. The structures of the isolated agonist binding domain of the three subtypes of glutamate receptors have provided the first insight into the conformational changes and mechanism by which the agonist could control the activation and desensitization of the channel (1-10). However, most of the structures are of the ␣-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA) subtype, for which currently there are Ͼ60 structures in various ligated states (1,8,11). There is also significant insight into the dynamic state of the agonist binding domain for the AMPA receptors and on the role of specific agonist-protein interactions (9,(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). These studies show that for a number of cases the agonist binding domains (ABDs) 3 of the AMPA receptors show a graded cleft closure with the extent of cleft closure correlating to the extent of activation (efficacy) of the agonist. Similar investigations on the kainate receptors also show a similar mechanism of activation. The NMDA subtype of the glutamate receptor, on the other hand, is the least studied in terms of structures (2,3,27). These receptors are different from the other subtypes as they are obligate heteromers of the glycine-binding GluN1 or GluN3 subunits and the glutamate-binding GluN2 (28 -30), whereas non-NMDA receptors such as AMPA and kainate receptors can form functional homotetrameric channels activated solely by glutamate. For the NMDA receptors, the structures of the agonist binding domain of the GluN1 subunit bound to full agonist glycine and partial agonists such as D-cycloserine showed no significant differences (2, 27). Based on these results, it has been suggested that the NMDA receptors co...

“…Gating could be a result of dynamic processes within S1S2 or in other parts of the protein. For example, backbone NMR dynamics measurements (17,29) have found that Lobe 2 is considerably more dynamic than Lobe 1, and this could be associated with channel gating. One specific possibility concerns motions near the linker region (i.e.…”

Section: Discussionmentioning

confidence: 99%

Dynamics of the S1S2 Glutamate Binding Domain of GluR2 Measured Using 19F NMR Spectroscopy

Ahmed

Loh

Jane

et al. 2007

Self Cite

Ionotropic glutamate receptors mediate the majority of vertebrate excitatory synaptic transmission. Although the structure of the GluR2 binding domain (S1S2) is well known (agonist binding site between two lobes), little is known about the time scales of conformational transitions or the relationship between dynamics and function.19 F NMR ( 19 F-labeled tryptophan) spectroscopy was used to monitor motions in the S1S2 domain bound to ligands with varying efficacy and in the apo state. One tryptophan (Trp-671) undergoes chemical exchange in some but not all agonists, consistent with s-ms motion. The dynamics can be correlated to ligand affinity, and a likely source of the motion is a peptide bond capable of transiently forming hydrogen bonds across the lobe interface. Another tryptophan (Trp-767) appears to monitor motions of the relative positions of the lobes and suggests that the relative orientation in the apo-and antagonist-bound forms can exchange between at least two conformations on the ms time scale. Ionotropic glutamate receptors (GluRs)2 mediate the majority of excitatory synaptic transmission in the central nervous system of higher vertebrates (1) and play important roles in the formation of synaptic plasticity underlying higher order processes such as learning and memory as well as in neuronal development (2). In addition, ionotropic GluRs have been implicated in various neurodegenerative disorders such as Parkinson and Alzheimer diseases, Huntington chorea, and neurologic disorders including epilepsy and ischemic brain damage. Antagonists of glutamate receptors have been shown to limit tumor growth in a variety of human tumors and to inhibit tumor cell migration (3). In recent years many advances in characterizing the relationship between ionotropic GluR structure and function have been made. Ionotropic GluRs are membrane-bound receptor ion channels composed of multiple subunits arranged as a rosette, forming a central ion channel in which each subunit contributes to pore formation. Individual subunits are categorized by pharmacological properties, sequence, functionality, and biological roles into those that are sensitive 1) to the synthetic agonist N-methyl-D-aspartic acid (NR1, NR2A-D, NR3A-B), 2) to the synthetic agonist ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA; GluR1-4), and 3) to the naturally occurring neurotoxin kainate (GluR5-7, KA1,2).The structural analysis of glutamate receptors was dramatically advanced by the finding that the extracellular agonist binding domain (S1S2 domain) could be expressed in isolation as a soluble protein (4) and by the subsequent solution of the crystal structure bound to kainate (5). As predicted by homology models (6), the S1S2 domain was found to be a bilobed structure with the agonist binding pocket located between the two lobes. The first glimpse of the structural basis of channel activation was the finding by Armstrong and Gouaux (7) that the apo state exhibited a considerably larger angle between the two lobes than the full agonist-bound...