Role of the chemical interactions of the agonist in controlling alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor activation

Alpha-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, to establish the role of specific molecular interactions in controlling fine details such 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 and Y450F and L650T mutants of the GluR2 subtype, and by using the multiscale investigation, we show that the strength of the interactions at the alpha-amine group of the agonist with the protein in all but one case tracks the extent of activation. Since the alpha-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.     

Chemistry and conformation of the ligand-binding domain of GluR2 subtype of glutamate receptors

In the present report, using vibrational spectroscopy we have probed the ligand-protein interactions for full agonists (glutamate and alpha-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 alpha-carboxylates for the various agonists, with kainate having the strongest interactions and glutamate having the weakest. Additionally, the interactions at the alpha-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 alpha-amine group. These investigations suggest that the interactions at the alpha-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 alpha-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.     

Conformational changes at the agonist binding domain of the N-methyl-D-aspartic acid receptor

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 α-amino-5-methyl-3-hydroxy-4-isoxazole propionate and kainate subtypes of the ionotropic glutamate receptors.     

Stereochemistry of glutamate receptor agonist efficacy: engineering a dual-specificity AMPA/kainate receptor

Upon agonist binding, the bilobate ligand-binding domains of the ionotropic glutamate receptors (iGluR) undergo a cleft closure whose magnitude correlates broadly with the efficacy of the agonist. AMPA (alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid) and kainate are nonphysiological agonists that distinguish between subsets of iGluR. Kainate acts with low efficacy at AMPA receptors. Here we report that the structure-based mutation L651V converts the GluR4 AMPA receptor into a dual-specificity AMPA/kainate receptor fully activated by both agonists. To probe the stereochemical basis of partial agonism, we have also investigated the correlation between agonist efficacy and a series of vibrational and fluorescence spectroscopic signals of agonist binding to the corresponding wild-type and mutant GluR4 ligand-binding domains. Two signals track the extent of channel activation: the maximal change in intrinsic tryptophan fluorescence and the environment of the single non-disulfide bonded C426, which appears to probe the strength of interactions with the ligand alpha-amino group. Both of these signals arise from functional groups that are poised to detect changes in the extent of channel cleft closure and thus provide additional information about the coupling between conformational changes in the ligand-binding domain and activation of the intact receptor.     

Luminescence resonance energy transfer investigation of conformational changes in the ligand binding domain of a kainate receptor

The apo state structure of the isolated ligand binding domain of the GluR6 subunit and the conformational changes induced by agonist binding to this protein have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances show that agonist binding induces cleft closure, and the extent of cleft closure is proportional to the extent of activation over a wide range of activations, thus establishing that the cleft closure conformational change is one of the mechanisms by which the agonist mediates receptor activation. The LRET distances also provide insight into the apo state structure, for which there is currently no crystal structure available. The distance change between the glutamate-bound state and the apo state is similar to that observed between the glutamate-bound and antagonist UBP-310-bound form of the GluR5 ligand binding domain, indicating that the cleft for the apo state of the GluR6 ligand binding domain should be similar to the UBP-310-bound form of GluR5. This observation implies that te apo state of GluR6 undergoes a cleft closure of 29-30 degrees upon binding full agonists, one of the largest observed in the glutamate receptor family.     

LRET investigations of conformational changes in the ligand binding domain of a functional AMPA receptor

The structural investigations using the soluble ligand binding domain of the AMPA subtype of the glutamate receptor have provided invaluable insight into the mechanistic pathway by which agonist binding to this extracellular domain mediates the formation of cation-selective channels in this protein. These structures, however, are in the absence of the transmembrane segments, the primary functional component of the protein. Here, we have used a modified luminescence resonance energy transfer based method to obtain distance changes due to agonist binding in the ligand binding domain in the presence of the transmembrane segments. These distance changes show that the cleft closure conformational change observed in the isolated ligand binding domain upon binding agonist is conserved in the receptor with the channel segments, thus establishing that the isolated ligand binding domain is a good model of the domain in the receptor containing the transmembrane segments.     

Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core

Crystal structures of the GluR2 ligand binding core (S1S2) have been determined in the apo state and in the presence of the antagonist DNQX, the partial agonist kainate, and the full agonists AMPA and glutamate. The domains of the S1S2 ligand binding core are expanded in the apo state and contract upon ligand binding with the extent of domain separation decreasing in the order of apo > DNQX > kainate > glutamate approximately equal to AMPA. These results suggest that agonist-induced domain closure gates the transmembrane channel and the extent of receptor activation depends upon the degree of domain closure. AMPA and glutamate also promote a 180 degrees flip of a trans peptide bond in the ligand binding site. The crystal packing of the ligand binding cores suggests modes for subunit-subunit contact in the intact receptor and mechanisms by which allosteric effectors modulate receptor activity.     

Crystal structures of the GluR5 and GluR6 ligand binding cores: molecular mechanisms underlying kainate receptor selectivity

Little is known about the molecular mechanisms underlying differences in the ligand binding properties of AMPA, kainate, and NMDA subtype glutamate receptors. Crystal structures of the GluR5 and GluR6 kainate receptor ligand binding cores in complexes with glutamate, 2S,4R-4-methylglutamate, kainate, and quisqualate have now been solved. The structures reveal that the ligand binding cavities are 40% (GluR5) and 16% (GluR6) larger than for GluR2. The binding of AMPA- and GluR5-selective agonists to GluR6 is prevented by steric occlusion, which also interferes with the high-affinity binding of 2S,4R-4-methylglutamate to AMPA receptors. Strikingly, the extent of domain closure produced by the GluR6 partial agonist kainate is only 3 degrees less than for glutamate and 11 degrees greater than for the GluR2 kainate complex. This, together with extensive interdomain contacts between domains 1 and 2 of GluR5 and GluR6, absent from AMPA receptors, likely contributes to the high stability of GluR5 and GluR6 kainate complexes.     

Dynamics of Cleft Closure of the GluA2 Ligand-binding Domain in the Presence of Full and Partial Agonists Revealed by Hydrogen-Deuterium Exchange

The majority of excitatory neurotransmission in the CNS is mediated by tetrameric AMPA receptors. Channel activation begins with a series of interactions with an agonist that binds to the cleft between the two lobes of the ligand-binding domain of each subunit. Binding leads to a series of conformational transitions, including the closure of the two lobes of the binding domain around the ligand, culminating in ion channel opening. Although a great deal has been learned from crystal structures, determining the molecular details of channel activation, deactivation, and desensitization requires measures of dynamics and stabilities of hydrogen bonds that stabilize cleft closure. The use of hydrogen-deuterium exchange at low pH provides a measure of the variation of stability of specific hydrogen bonds among agonists of different efficacy. Here, we used NMR measurements of hydrogen-deuterium exchange to determine the stability of hydrogen bonds in the GluA2 (AMPA receptor) ligand-binding domain in the presence of several full and partial agonists. The results suggest that the stabilization of hydrogen bonds between the two lobes of the binding domain is weaker for partial than for full agonists, and efficacy is correlated with the stability of these hydrogen bonds. The closure of the lobes around the agonists leads to a destabilization of the hydrogen bonding in another portion of the lobe interface, and removing an electrostatic interaction in Lobe 2 can relieve the strain. These results provide new details of transitions in the binding domain that are associated with channel activation and desensitization.     

Molecular mechanism of ligand recognition by NR3 subtype glutamate receptors

NR3 subtype glutamate receptors have a unique developmental expression profile, but are the least well-characterized members of the NMDA receptor gene family, which have key roles in synaptic plasticity and brain development. Using ligand binding assays, crystallographic analysis, and all atom MD simulations, we investigate mechanisms underlying the binding by NR3A and NR3B of glycine and D-serine, which are candidate neurotransmitters for NMDA receptors containing NR3 subunits. The ligand binding domains of both NR3 subunits adopt a similar extent of domain closure as found in the corresponding NR1 complexes, but have a unique loop 1 structure distinct from that in all other glutamate receptor ion channels. Within their ligand binding pockets, NR3A and NR3B have strikingly different hydrogen bonding networks and solvent structures from those found in NR1, and fail to undergo a conformational rearrangement observed in NR1 upon binding the partial agonist ACPC. MD simulations revealed numerous interdomain contacts, which stabilize the agonist-bound closed-cleft conformation, and a novel twisting motion for the loop 1 helix that is unique in NR3 subunits.     

Vibrational spectroscopic investigation of the ligand binding domain of kainate receptors

Fourier transform infrared spectroscopy has been used to probe the agonist‐protein interactions in the ligand binding domain of the GluR6 subunit, one subunit of the kainate subtype of glutamate receptors. In order to study the changes in the interactions over a range of activations the investigations were performed using the wild type, N690S, and T661E mutations. These studies show that the strength of the interactions at the α‐amine group of the agonist, as probed by studying the environment of the nondisulphide bonded Cys 432, acts as a switch with weaker interactions at lower activations and stronger interactions at higher activations. The α‐carboxylate interactions of the agonist, however, are not significantly different over the wide range of activations, as measured by the maximum currents mediated by the receptors at saturating concentrations of agonists. Previous investigations of AMPA receptors show a similar dependence of the α‐amine interactions on activation indicating that the roles of the α‐amine interactions in mediating receptor activation are similar for both subtypes of receptors; however, in the case of the AMPA receptors a tug of war type of change was observed between the α‐amine and α‐carboxylate interactions and this is not observed in kainate receptors. This decoupling of the two interactions could arise due to the larger cleft observed in kainate receptors, which allows for a more flexible interaction for the α‐amine and α‐carboxylate groups of the agonists.     

Disruption of interdomain interactions in the glutamate binding pocket affects differentially agonist affinity and efficacy of N-methyl-D-aspartate receptor activation

In ionotropic glutamate receptors, agonist binding occurs in a conserved clam shell-like domain composed of the two lobes D1 and D2. Docking of glutamate into the binding cleft promotes rotation in the hinge region of the two lobes, resulting in closure of the binding pocket, which is thought to represent a prerequisite for channel gating. Here, we disrupted D1D2 interlobe interactions in the NR2A subunit of N-methyl-d-aspartate (NMDA) receptors through systematic mutation of individual residues and studied the influence on the activation kinetics of currents from NR1/NR2 NMDA receptors heterologously expressed in HEK cells. We show that the mutations affect differentially glutamate binding and channel gating, depending on their location within the binding domain, mainly by altering k(off) and k(cl), respectively. Whereas impaired stability of glutamate in its binding site is the only effect of mutations on one side of the ligand binding pocket, close to the hinge region, alterations in gating are the predominant consequence of mutations on the opposite side, at the entrance of the binding pocket. A mutation increasing D1D2 interaction at the entrance of the pocket resulted in an NMDA receptor with an increased open probability as demonstrated by single channel and whole cell kinetic analysis. Thus, the results indicate that agonist-induced binding domain closure is itself a complex process, certain aspects of which are coupled either to binding or to gating. Specifically, we propose that late steps of domain closure, in kinetic terms, represent part of channel gating.     

The loss of an electrostatic contact unique to AMPA receptor ligand binding domain 2 slows channel activation

Ligand-gated ion channels undergo conformational changes that transfer the energy of agonist binding to channel opening. Within ionotropic glutamate receptor (iGluR) subunits, this process is initiated in their bilobate ligand binding domain (LBD) where agonist binding to lobe 1 favors closure of lobe 2 around the agonist and allows formation of interlobe hydrogen bonds. AMPA receptors (GluAs) differ from other iGluRs because glutamate binding causes an aspartate-serine peptide bond in a flexible part of lobe 2 to rotate 180° (flipped conformation), allowing these residues to form cross-cleft H-bonds with tyrosine and glycine in lobe 1. This aspartate also contacts the side chain of a lysine residue in the hydrophobic core of lobe 2 by a salt bridge. We investigated how the peptide flip and electrostatic contact (D655-K660) in GluA3 contribute to receptor function by examining pharmacological and structural properties with an antagonist (CNQX), a partial agonist (kainate), and two full agonists (glutamate and quisqualate) in the wildtype and two mutant receptors. Alanine substitution decreased the agonist potency of GluA3(i)-D655A and GluA3(i)-K660A receptor channels expressed in HEK293 cells and differentially affected agonist binding affinity for isolated LBDs without changing CNQX affinity. Correlations observed in the crystal structures of the mutant LBDs included the loss of the D655-K660 electrostatic contact, agonist-dependent differences in lobe 1 and lobe 2 closure, and unflipped D(A)655-S656 bonds. Glutamate-stimulated activation was slower for both mutants, suggesting that efficient energy transfer of agonist binding within the LBD of AMPA receptors requires an intact tether between the flexible peptide flip domain and the rigid hydrophobic core of lobe 2.     

The structural basis for kainoid selectivity at AMPA receptors revealed by low-mode docking calculations

The kainoids are a class of excitatory and excitotoxic pyrrolidine dicarboxylates that act at ionotropic glutamate receptors. The kainoids bind kainate receptors with high affinity and, while binding affinity is lower at AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, they are active in functional assays at this receptor subtype as well. However, kainoids are only partial agonists at AMPA receptors. Currents evoked by kainoids have been described as either slowly desensitizing, partially desensitizing, or non-desensitizing. Recently acquired X-ray crystal structures of the ligand binding domain of the iGluR2, AMPA sensitive receptor suggest that differences in ligand-receptor interactions may influence functional properties of an agonist. In an effort to identify important ligand-receptor interactions of various kainoids, we have conducted a series of low-mode docking searches of AMPA agonists in the iGluR2 binding domain. Kainic acid exhibited alternate low-lying geometries, with loss of hydrogen bonds to domain 2, which may represent a dissociation route not available to other kainoids. The most potent of the kainoids are capable of forming hydrogen bonding interactions that span the two domains of the receptor. In particular, a hydrogen bond between the domoic acid C6' carboxylic acid and Ser652 may prevent a peptide bond rotation that is associated with the desensitized state of the receptor.     

Mechanisms of activation,inhibition and specificity: crystal structures of the NMDA receptor NR1 ligand-binding core

Excitatory neurotransmission mediated by the N-methyl-D-aspartate subtype of ionotropic glutamate receptors is fundamental to the development and function of the mammalian central nervous system. NMDA receptors require both glycine and glutamate for activation with NR1 and NR2 forming glycine and glutamate sites, respectively. Mechanisms to describe agonist and antagonist binding, and activation and desensitization of NMDA receptors have been hampered by the lack of high-resolution structures. Here, we describe the cocrystal structures of the NR1 S1S2 ligand-binding core with the agonists glycine and D-serine (DS), the partial agonist D-cycloserine (DCS) and the antagonist 5,7-dichlorokynurenic acid (DCKA). The cleft of the S1S2 'clamshell' is open in the presence of the antagonist DCKA and closed in the glycine, DS and DCS complexes. In addition, the NR1 S1S2 structure reveals the fold and interactions of loop 1, a cysteine-rich region implicated in intersubunit allostery.     

A Charge-inverting Mutation in the “Linker” Region of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Alters Agonist Binding and Gating Kinetics Independently of Allosteric Modulators

AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728–4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.     

Conformational changes in the ligand-binding domain of a functional ionotropic glutamate receptor

Fluorescence resonance energy transfer was used to determine the structural changes in the extracellular ligand-binding segment in a functional glutamate receptor that contains the ligand-binding, transmembrane, and C-terminal segments. These studies indicate that the structural changes previously reported for the isolated ligand-binding domain due to the binding of partial and full agonists are also observed in this functional receptor, thus validating the detailed structure-function relationships that have been previously developed based on the structure of the isolated ligand-binding domain. Additionally, these studies provide the first evidence that there are no significant changes in the extent of cleft closure between the activated and desensitized states of the glutamate bound form of the receptor consistent with the previous functional investigations, which suggest that desensitization is mediated primarily by changes in the interactions between subunits composing the receptor.     

Interplay between structural rigidity and electrostatic interactions in the ligand binding domain of GluR2

Using molecular dynamics (MD) simulations, computational protein modifications, and a novel theoretical methodology that determines structural rigidity/flexibility (the FIRST algorithm), we investigate how molecular structure and dynamics of the glutamate receptor ligand binding domain (GluR2 S1S2) facilitate its conformational transition. S1S2 is a two-lobe protein, which undergoes a cleft closure conformational transition upon binding an agonist in the cleft between the two lobes; hence it is expected that the mechanism of this conformational transition can be characterized as a hinge-type. However, in the rigidity analysis one lobe of the protein is identified as a single rigid cluster while the other one is structurally flexible, inconsistent with a presumed mechanical hinge mechanism. Instead, we characterize the cleft-closing transition as a load and lock mechanism. We find that when two cross-cleft hydrogen bonds are disrupted the protein undergoes a rapid cleft opening transition. At the same time, the dynamical behavior of the cleft in the presence of the glutamate ligand is only weakly affected by the S652 peptide bond in its flipped conformation observed in the crystal structure. The residue E705 plays significant role in stabilization of the closed conformation via electrostatic interactions. The presence of the E705-K730 salt bridge seems to correlate strongly withthe cleft opening transition in the MD simulations.     

On the binding determinants of the glutamate agonist with the glutamate receptor ligand binding domain

Ionotropic glutamate receptors (GluRs) are ligand-gated membrane channel proteins found in the central neural system that mediate a fast excitatory response of neurons. In this paper, we report theoretical analysis of the ligand-protein interactions in the binding pocket of the S1S2 (ligand binding) domain of the GluR2 receptor in the closed conformation. By utilizing several theoretical methods ranging from continuum electrostatics to all-atom molecular dynamics simulations and quantum chemical calculations, we were able to characterize in detail glutamate agonist binding to the wild-type and E705D mutant proteins. A theoretical model of the protein-ligand interactions is validated via direct comparison of theoretical and Fourier transform infrared spectroscopy (FTIR) measured frequency shifts of the ligand's carboxylate group vibrations [Jayaraman et al. (2000) Biochemistry 39, 8693-8697; Cheng et al. (2002) Biochemistry 41, 1602-1608]. A detailed picture of the interactions in the binding site is inferred by analyzing contributions to vibrational frequencies produced by protein residues forming the ligand-binding pocket. The role of mobility and hydrogen-bonding network of water in the ligand-binding pocket and the contribution of protein residues exposed in the binding pocket to the binding and selectivity of the ligand are discussed. It is demonstrated that the molecular surface of the protein in the ligand-free state has mainly positive electrostatic potential attractive to the negatively charged ligand, and the potential produced by the protein in the ligand-binding pocket in the closed state is complementary to the distribution of the electrostatic potential produced by the ligand itself. Such charge complementarity ensures specificity to the unique charge distribution of the ligand.