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.
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.
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 ␣-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. N-Methyl-D-aspartate (NMDA)3 receptors are members of the ionotropic glutamate receptor channel family, which mediate most of the fast excitatory synaptic transmissions in the central nervous system. The glutamate receptor family consists of three subtypes: kainate receptors, ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, and NMDA receptors (2-4). Although the overall topology is similar among the three subtypes of the glutamate receptors, the NMDA receptors differ in many ways from the AMPA and kainate receptor subtypes. Functionally, the NMDA receptors are highly permeable to calcium ions and exhibit voltage-dependent block by physiological concentrations of Mg 2ϩ and act as a coincidence detector, permitting ion flow only upon depolarization of the receiving cell. Structurally, unlike the AMPA and kainate receptors that can form homomeric receptors, the NMDA receptors are obligate heteromeric receptors composed of homologous NR1, NR2, and/or NR3 subunits.A large fraction of the neuronal NMDA receptors are composed of glycine binding NR1 and glutamate binding NR2 subunits (5-8). The structure of the isolated ligand binding domain of the NMDA receptor suggests that the tetrameric receptor has a dimer of dimer arrangement, with each dimer having one glycine binding subunit and one glutamate binding subunit (9). The recent full-length structure of the AMPA receptor published while this manuscript was in review along with cross-linking data suggest that the dimers are arranged such that the NR1 subunits are diagonal to each other, as are...
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...
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