Solution X-ray Scattering Evidence for Agonist- and Antagonist-induced Modulation of Cleft Closure in a Glutamate Receptor Ligand-binding Domain

Madden, Dean R.; Armstrong, N.; Svergun, Dmitri I.; Pérez, Javier; Vachette, Patrice

doi:10.1074/jbc.m414523200

Cited by 25 publications

(28 citation statements)

References 35 publications

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“…We consider three conformational states of LBD: ''apo'' or unliganded state (PDB entry: 1FTO), antagonist-bound state (PDB entry: 1FTL), and partial agonist-bound state (PDB entry: 1FTK). It is experimentally shown that the conformation of ''apo'' state is more similar to the antagonist-bound state than to the agonist-bound state (Madden et al, 2005). As we will see shortly, the same conclusion can be drawn from our simulation results.…”

Section: Glutamate Receptor Lbd Conformationssupporting

confidence: 77%

Cross-Validation of Data Compatibility Between Small Angle X-ray Scattering and Cryo-Electron Microscopy

Kim

Afsari

Chirikjian

2017

Journal of Computational Biology

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Cryo-electron microscopy (EM) and small angle X-ray scattering (SAXS) are two different data acquisition modalities often used to glean information about the structure of large biomolecular complexes in their native states. A SAXS experiment is generally considered fast and easy but unveils the structure at very low resolution, whereas a cryo-EM experiment needs more extensive preparation and postacquisition computation to yield a threedimensional (3D) density map at higher resolution. In certain applications, we may need to verify whether the data acquired in the SAXS and cryo-EM experiments correspond to the same structure (e.g., before reconstructing the 3D density map in EM). In this article, a simple and fast method is proposed to verify the compatibility of the SAXS and EM experimental data. The method is based on averaging the two-dimensional correlation of EM images and the Abel transform of the SAXS data. Orientational preferences are known to exist in cryo-EM experiments, and we also consider these effects on our method. The results are verified on simulations of conformational states of large biomolecular complexes.

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Section: Glutamate Receptor Lbd Conformationssupporting

confidence: 77%

Cross-Validation of Data Compatibility Between Small Angle X-ray Scattering and Cryo-Electron Microscopy

Kim

Afsari

Chirikjian

2017

Journal of Computational Biology

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“…The smaller changes observed upon going from the apo to the ligated state in the FRET lifetimes are consistent with the small-angle scattering results. The scattering data for the apo state can be fit by a structure similar to the kainate-bound state (more cleft closure) or by an equilibrium of the apo and fully ligated crystal states (10). A kainate-like structure for the apo state would be consistent with the smaller changes in the distance between the apo and AMPA-bound forms observed in the FRET experiments described here.…”

Section: Resultsmentioning

confidence: 67%

Allosteric mechanism in AMPA receptors: A FRET-based investigation of conformational changes

Ramanoudjame¹,

Du²,

Mankiewicz³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the primary mediators of fast excitatory synaptic transmission in the mammalian CNS. Structures of the extracellular ligandbinding domain suggest that the extent of cleft closure in the ligand-binding domain controls the extent of activation of the receptor. Here we have developed a fluorescence resonance energy transfer-based probe that allows us to study the extent of cleft closure in the isolated ligand-binding domain in solution. These investigations show that the wild-type protein exhibits a graded cleft closure that correlates to the extent of activation, which is in qualitative agreement with the crystal structures. However, the changes in extent of cleft closure between the apo and agonist-bound states are smaller than that observed in the crystal structures. We have also used this method to study the L650T mutant and show that in solution the ␣-amino-5-methyl-3-hydroxy-4-isoxazole propionate-bound form of this mutant exists primarily in a conformation that is more closed than predicted based on the activity, indicating that the degree of cleft closure alone cannot be used as a measure of extent of activation of the receptor, and there are possibly other mechanisms in addition to cleft closure that mediate the subtleties in extent of activation by a given agonist.fluorescence ͉ glutamate ͉ ion channel L igand-gated ion channels are allosteric proteins that convert chemical signals into electrical signals by forming transmembrane ion channels upon ligand binding to an extracellular domain. Ionotropic glutamate receptors, a member of the ligand-gated ion channels, serve as an excellent paradigm for studying allostery in this family of proteins (1-6). The crystal structures of the isolated ligand-binding domain of the GluR2 subunit of the ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor protein show a graded cleft closure with a direct correlation between the extent of cleft closure induced by a ligand and its extent of activation (5, 6), which is consistent with a multiple state-induced fit model. However, there is one exception to this correlation, the AMPA-bound form of the L650T mutation of the receptor crystallizes in two forms: one structure where the cleft is closed 11°and a second structure in which the cleft is closed 22°relative to the open apo form of the protein (7). These structures lie on either side of the cleft closure versus activation correlation observed for the wild-type protein, thus raising the question as to whether some ligands or modifications at the AMPA receptors can change the mechanism of activation from the multistate model to that defined by equilibrium of two distinct states.Here we have developed a fluorescence resonance energy transfer (FRET)-based assay that allows us to investigate the conformational changes in the ligand-binding domain in solution, thus allowing us to probe the conformational changes in this domain without the crystallographic constraints. In addition, we have u...

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“…Furthermore, it may be difficult to identify and correct for such discrepancies when ⌬G E corresponds to local backbone unfolding events. However, because the key H N s considered in the present study presumably become solvent-exposed upon ligand detachment or domain opening, it is possible in principle to estimate their k HDX from experiments employing either the (38), the rapid docking of agonist to the protein (20,21) argues against explanation (i). Similar arguments against extended closed states in the Apo protein can be made from free energy calculations as a function of the degree of lobe closure (39,40).…”

Section: Keymentioning

confidence: 91%

On the Mechanisms of α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) Receptor Binding to Glutamate and Kainate

Fenwick

Oswald

2010

Journal of Biological Chemistry

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The ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of ionotropic glutamate receptors mediates much of the fast excitatory neurotransmission in the central nervous system. The ability of these receptors to shape such responses appears to be due in part to dynamic processes induced by agonists in the ligand-binding domain. Previous studies employing fluorescence spectroscopy and whole cell recording suggest that agonist binding is followed by sequential transitions to one or more distinct conformational states. Here, we used hydrogen-deuterium exchange to determine the mechanisms of binding of glutamate and kainate (full and partial agonists, respectively) to a soluble ligand-binding domain of GluR2. Our results provide a structural basis for sequential state models of agonist binding and the free energy changes of the associated state-to-state transitions. For glutamate, a multi-equilibrium binding reaction was discerned involving distinct ligand docking, domain isomerization, and lobe-locking steps. In contrast, kainate binding involves a simpler dock-isomerization process in which the isomerization equilibrium is shifted dramatically toward open domain conformations. In light of increasing evidence that the stability, in addition to the extent, of domain closure is a critical component of the channel activation mechanism, the differences in domain opening and closing equilibria detected for glutamate and kainate should be useful structural measures for interpreting the markedly different current responses evoked by these agonists.As a consequence of glutamate binding, AMPA 2 receptors produce a rapid flux of cations into post-synaptic neurons that is vital for information transfer in the central nervous system. In addition, these receptors undergo fast deactivation and desensitization along with slower recovery kinetics, which together shape the time course of current decay and restoration (1-6). Structurally, AMPA receptors are tetrameric membranebound proteins composed of modular subunits having two large extracellular domains, a membrane-spanning ion channel, and a C-terminal intracellular region (4, 7-12). One of the extracellular domains, S1S2, is a bilobed structure that binds agonists in a cleft between its lobes (13). Domain closure of S1S2 around the agonist leads to channel opening, and maximum currents have been attributed to complete lobe closure upon complex formation with full agonists (13-17). However, among the full agonists examined, those like glutamate, which are less potent and which bind to S1S2 with lower affinity, produce higher rates of channel deactivation and resensitization (18), despite inducing the same degree of cleft closure in S1S2. This suggests that AMPA receptor function is dependent not only on the extent of lobe closure caused by the agonist but also on the lobe closing and opening dynamics associated with agonist binding and dissociation (19).A prevailing model for the binding reaction is

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Solution X-ray Scattering Evidence for Agonist- and Antagonist-induced Modulation of Cleft Closure in a Glutamate Receptor Ligand-binding Domain

Cited by 25 publications

References 35 publications

Cross-Validation of Data Compatibility Between Small Angle X-ray Scattering and Cryo-Electron Microscopy

Cross-Validation of Data Compatibility Between Small Angle X-ray Scattering and Cryo-Electron Microscopy

Allosteric mechanism in AMPA receptors: A FRET-based investigation of conformational changes

On the Mechanisms of α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) Receptor Binding to Glutamate and Kainate

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