Role of conformational dynamics in α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor partial agonism.

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.

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

AMPA receptors mediate fast excitatory neurotransmission by converting chemical signals into electrical signals, and thus it is important to understand the relationship between their chemical biology and their function. We used single-molecule fluorescence resonance energy transfer to examine the conformations explored by the agonist-binding domain of the AMPA receptor for wild-type and T686S mutant proteins. Each form of the agonist binding domain showed a dynamic, multistate sequential equilibrium, which could be identified only using wavelet shrinkage, a signal processing technique that removes experimental shot noise. These results illustrate that the extent of activation depends 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 determined not only by the lowest energy state but also by the range of states that the protein explores.

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.

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 alpha-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.

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.

A natural human monoclonal antibody targeting Staphylococcus Protein A protects against Staphylococcus aureus bacteremia.

Staphylococcus aureus can cause devastating and life-threatening infections. With the increase in multidrug resistant strains, novel therapies are needed. Limited success with active and passive immunization strategies have been attributed to S. aureus immune evasion. Here, we report on a monoclonal antibody, 514G3, that circumvents a key S. aureus evasion mechanism by targeting the cell wall moiety Protein A (SpA). SpA tightly binds most subclasses of immunoglobulins via their Fc region, neutralizing effector function. The organism can thus shield itself with a protective coat of serum antibodies and render humoral immunity ineffective. The present antibody reactivity was derived from an individual with natural anti-SpA antibody titers. The monoclonal antibody is of an IgG3 subclass, which differs critically from other immunoglobulin subclasses since its Fc is not bound by SpA. Moreover, it targets a unique epitope on SpA that allows it to bind in the presence of serum antibodies. Consequently, the antibody opsonizes S. aureus and maintains effector function to enable natural immune mediated clearance. The data presented here provide evidence that 514G3 antibody is able to successfully rescue mice from S. aureus mediated bacteremia.

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 alpha-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 alpha-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 alpha-amine interactions on activation indicating that the roles of the alpha-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 alpha-amine and alpha-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 alpha-amine and alpha-carboxylate groups of the agonists.

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.

Chemical interplay in the mechanism of partial agonist activation in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, one subtype in the family of ionotropic glutamate receptors, are the main receptors responsible for excitatory signaling in the mammalian central nervous system. Previous studies utilitizing the isolated ligand binding domain of these receptors have provided insight into the role of specific ligand-protein interactions in mediating receptor activation. However, these studies relied heavily on the partial agonist kainate, in which the alpha-amine group is constrained in a pyrrolidine ring. Here we have studied a series of substituted and unsubstituted willardiines with primary alpha-amine groups similar to that of the full agonist glutamate whose activation can be varied depending on the size of the substituent. The specific ligand-protein interactions in the mechanism of partial agonism in this subtype were investigated using vibrational spectroscopy, and the large-scale conformational changes in the ligand binding domain were studied with fluorescence resonance energy transfer (FRET). These investigations show that the strength of the interaction at the alpha-amine group correlates with the extent of cleft closure and extent of activation, with the agonist of higher efficacy showing larger cleft closure and stronger interactions at this group, suggesting that this is one of the mechanisms by which the agonist controls receptor activation.

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.