Anu Rambhadran

Structural landscape of isolated agonist-binding domains from single AMPA receptors

α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors mediate fast excitatory neurotransmission by converting chemical signals into electrical signals. Thus, it is important to understand the relationship between their chemical biology and their function. Single molecule fluorescence resonance energy transfer (smFRET) was used to examine the conformations explored by the agonist binding domain of the AMPA receptor for wild type and T686 mutant proteins. Each form of the agonist binding domain exhibited a dynamic, multi-state sequential equilibrium, which could only be identified using wavelet shrinkage, a signal processing technique that removes experimental shot-noise. These results illustrate that the extent of activation is dependent not on a rigid closed cleft, but instead on the probability that a given subunit will occupy a closed cleft conformation, which in turn is not only determined by the lowest energy state but by the range of states that the protein explores.

Subunit arrangement in N-methyl-D-aspartate (NMDA) receptors.

N-Methyl-d-aspartate (NMDA) receptors, the main mediators of excitatory synaptic transmission, are heterotetrameric receptors. Typically, glycine binding NR1 subunits co-assemble with glutamate binding NR2 subunits to form a functional receptor. Here we have used luminescence resonance energy transfer (LRET) investigations to establish the specific configuration in which these subunits assemble to form the functional tetramer and show that the dimer of dimers structure is formed by the NR1 subunits assembling diagonally to each other. The distances measured by LRET are consistent with the NMDA structure predicted based on cross-linking investigations and on the structure of the full-length α-amino-5-methyl-3-hydroxy-4-isoxazole propionic acid (AMPA) receptor structure (1). Additionally, the LRET distances between the NR1 and NR2A subunits within a dimer measured in the desensitized state of the receptor are longer than the distances in the previously published crystal structure of the isolated ligand binding domain of NR1-NR2A. Because the dimer interface in the isolated ligand binding domain crystallizes in the open channel structure, the longer LRET distances would be consistent with the decoupling of the dimer interface in the desensitized state. This is similar to what has been previously observed for the AMPA subtype of the ionotropic glutamate receptors, suggesting a similar mechanism for desensitization in the two subtypes of the glutamate receptor.

Role of Conformational Dynamics in α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) Receptor Partial Agonism

Background: Agonist binds to an extracellular agonist-binding domain in AMPA receptors. Results: Willardiines induce a range of cleft closure states in the agonist-binding domain of AMPA receptors. Conclusion: The fraction of the agonist-binding domains in a closed cleft conformation correlates with the extent of activation. Significance: The dynamics and extent of cleft closure in the agonist-binding domain control activation of AMPA receptors. We have investigated the range of cleft closure conformational states that the agonist-binding domains of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors occupy when bound to a series of willardiine derivatives using single-molecule FRET. These studies show that the agonist-binding domain exhibits varying degrees of dynamics when bound to the different willardiines with differing efficacies. The chlorowillardiine- and nitrowillardiine-bound form of the agonist-binding domain probes a narrower range of cleft closure states relative to the iodowillardiine bound form of the protein, with the antagonist (αS)-α-amino-3-[(4-carboxyphenyl)methyl]-3,4-dihydro-2,4-dioxo-1(2H)-pyrimidinepropanoic acid (UBP-282)-bound form exhibiting the widest range of cleft closure states. Additionally, the average cleft closure follows the order UBP-282 > iodowillardiine > chlorowillardiine > nitrowillardiine-bound forms of agonist-binding domain. These single-molecule FRET data, along with our previously reported data for the glutamate-bound forms of wild type and T686S mutant proteins, show that the mean currents under nondesensitizing conditions can be directly correlated to the fraction of the agonist-binding domains in the “closed” cleft conformation. These results indicate that channel opening in the AMPA receptors is controlled by both the ability of the agonist to induce cleft closure and the dynamics of the agonist-binding domain when bound to the agonist.

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.

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.

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° upon binding full agonists, one of the largest observed in the glutamate receptor family.

Mechanism of Activation in NMDA Receptors

The N-methyl D-aspartate receptors belong to the family of ionotropic glutamate receptors. They are obligate heteromeric receptors with two glycine binding subunits (GluN1) and two glutamate binding (GluN2A-D) subunits arranged as a dimer of dimers. We have used luminescence resonance energy transfer measurements to determine the specific arrangement of the GluN1 and GluN2 subunits within the tetramer. Based on this arrangement we have designed sites in the agonist binding domain that can probe changes in distances within the subunit and not across the subunits. By tagging these sites with donor and acceptor fluorophores we have investigated the conformational changes in the agonist binding domain of the GluN1 and GluN2A subunits using luminescence resonance energy transfer. 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. Single molecule FRET investigations are being performed to determine if the partial agonist bound state exhibits more flexibility relative to the full agonist bound state, accounting for the lower activation. The LRET-based distances for the glutamate binding GluN2A subunit, on the other hand, shows a graded cleft closure upon binding agonists with varying efficacy, with the extent of cleft closure being proportional to the extent of activation. This graded cleft closure indicates 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.

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.

NMDA Receptor Subunit Arrangement Probed By LRET

NMDA receptors are unique among the different subtypes of the ionotropic glutamate receptors since they require two agonists, glycine and glutamate, to bind to the receptor for the channel to open. They are heteromeric receptors composed of glycine binding subunits, such as NR1 and glutamate binding subunits, such as NR2, with the NR1 and NR2 subunits forming a dimer of dimers. While the crystal structures of the isolated ligand binding domain suggest that the dimer consists of one NR1 and one NR2 subunit, the arrangement of the dimer of dimers is not known, i.e. are the NR1 subunits adjacent, or across in the dimer of dimers. Using a modified NMDA receptor with specific donor:acceptor fluorophores at the N-terminus, and at various sites on the domain 1 of the ligand binding domain we have measured the distances between the NR1 subunits, between the NR2 subunits, and between NR1 and NR2 subunits, using luminescence resonance energy transfer. Using these distance constraints we show that the NR1-NR1 subunits and the NR2-NR2 subunits are adjacent to each other in forming the dimer of dimers.