Structural Dynamics of the Glycine-binding Domain of the N-Methyl-d-Aspartate Receptor

Dolino, Drew M.; Cooper, David R.; Ramaswamy, Swarna S.; Jaurich, Henriette; Landes, Christy F.; Jayaraman, Vasanthi

doi:10.1074/jbc.m114.605436

Cited by 37 publications

(45 citation statements)

References 31 publications

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“…First, the resulting smFRET distribution for the NMDAR LBD correlates well to the derived energy landscape seen in simulation studies of the glycine-bound NMDAR LBD (38,49), which, along with another recent smFRET study (37), supports the conclusion that we are indeed monitoring LBD cleft conformational states. More importantly, we introduce a model-free step transition and state identification method (STaSI) to identify additional cleft states in the isolated LBD that are too wide to be observed in the full hetero-tetramer.…”

Section: Introductionsupporting

confidence: 80%

“…Although it is unclear how these long-lived states relate to channel function in the full receptor, the states identified by smFRET correlate well to the broad distribution of states identified in umbrella sampling models of the LBD cleft in both NMDA and AMPA receptors (38,54). In addition, it is possible with both the AMPAR and NMDAR LBDs to relate average smFRET values for a variety of agonist conditions to agonist binding strength and macroscale ion transport across the channel (35)(36)(37).…”

Section: Introductionmentioning

confidence: 58%

“…1, inset) (24,25), which in turn opens the ion channel in the full-length protein (24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34). The average distance of the isolated LBD cleft closure has been shown to directly correlate to channel transport efficiency for multiple glutamate receptor types (18,(35)(36)(37), allowing the isolated LBD to be used as a model for further conformational analysis and prediction of channel transport efficiencies.…”

Section: Introductionmentioning

confidence: 99%

“…By collecting a statistically meaningful distribution of smFRET time trajectories, the entire ensemble equilibrium smFRET distribution, and therefore a detailed understanding of equilibrium dynamics, can be acquired (20,(34)(35)(36)(37)(38)46). Here, we apply smFRET to record the dynamics occurring in the LBD cleft ( Fig.…”

Section: Introductionmentioning

confidence: 99%

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Conformational Transitions in the Glycine-Bound GluN1 NMDA Receptor LBD via Single-Molecule FRET

Cooper

Dolino

Jaurich

et al. 2015

Biophysical Journal

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The N-methyl-D-aspartate receptor (NMDAR) is a member of the glutamate receptor family of proteins and is responsible for excitatory transmission. Activation of the receptor is thought to be controlled by conformational changes in the ligand binding domain (LBD); however, glutamate receptor LBDs can occupy multiple conformations even in the activated form. This work probes equilibrium transitions among NMDAR LBD conformations by monitoring the distance across the glycine-bound LBD cleft using single-molecule Förster resonance energy transfer (smFRET). Recent improvements in photoprotection solutions allowed us to monitor transitions among the multiple conformations. Also, we applied a recently developed model-free algorithm called "step transition and state identification" to identify the number of states, their smFRET efficiencies, and their interstate kinetics. Reversible interstate conversions, corresponding to transitions among a wide range of cleft widths, were identified in the glycine-bound LBD, on much longer timescales compared to channel opening. These transitions were confirmed to be equilibrium in nature by shifting the distribution reversibly via denaturant. We found that the NMDAR LBD proceeds primarily from one adjacent smFRET state to the next under equilibrium conditions, consistent with a cleft-opening/closing mechanism. Overall, by analyzing the state-to-state transition dynamics and distributions, we achieve insight into specifics of long-lived LBD equilibrium structural dynamics, as well as obtain a more general description of equilibrium folding/unfolding in a conformationally dynamic protein. The relationship between such long-lived LBD dynamics and channel function in the full receptor remains an open and interesting question.

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Section: Introductionsupporting

confidence: 80%

Section: Introductionmentioning

confidence: 58%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Conformational Transitions in the Glycine-Bound GluN1 NMDA Receptor LBD via Single-Molecule FRET

Cooper

Dolino

Jaurich

et al. 2015

Biophysical Journal

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“…4A). Umbrella sampling using an analogous order parameter was used previously to characterize conformational transitions in AMPA and NMDA receptor LBDs, and the results were found to be consistent with small-angle X-ray scattering (SAXS), ligand-binding affinity, and single-molecule FRET (smFRET) studies (17)(18)(19)(20)(21). Because in the crystal structures the side chains of Glu423 and Asp497 are in close proximity, pK a calculations were carried out using PROPKA (22) to determine which protonation states of these two residues to use in our simulations.…”

Section: Resultsmentioning

confidence: 59%

Molecular lock regulates binding of glycine to a primitive NMDA receptor

Alberstein

Thomas

et al. 2016

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

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The earliest metazoan ancestors of humans include the ctenophore Mnemiopsis leidyi. The genome of this comb jelly encodes homologs of vertebrate ionotropic glutamate receptors (iGluRs) that are distantly related to glycine-activated NMDA receptors and that bind glycine with unusually high affinity. Using ligandbinding domain (LBD) mutants for electrophysiological analysis, we demonstrate that perturbing a ctenophore-specific interdomain Arg-Glu salt bridge that is notably absent from vertebrate AMPA, kainate, and NMDA iGluRs greatly increases the rate of recovery from desensitization, while biochemical analysis reveals a large decrease in affinity for glycine. X-ray crystallographic analysis details rearrangements in the binding pocket stemming from the mutations, and molecular dynamics simulations suggest that the interdomain salt bridge acts as a steric barrier regulating ligand binding and that the free energy required to access open conformations in the glycine-bound LBD is largely responsible for differences in ligand affinity among the LBD variants.glutamate receptors | X-ray crystallography | electrophysiology | molecular dynamics simulations | free energy calculations

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Dual Unnatural Amino Acid Incorporation and Click‐Chemistry Labeling to Enable Single‐Molecule FRET Studies of p97 Folding

et al. 2016

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Many cellular functions are critically dependent on the folding of complex multimeric proteins, such as p97, a hexameric multi-domain AAA+ chaperone. Given p97’s complex architecture, single-molecule Förster Resonance Energy Transfer (smFRET) would be a powerful tool for studying folding while avoiding ensemble averaging. However, dual site-specific labeling of such a large protein for smFRET is a significant challenge. Here, we address this issue by using bioorthogonal azide-alkyne chemistry to attach an smFRET dye-pair to site-specifically incorporate unnatural amino acids, allowing us to generate p97 variants reporting on inter- or intra-domain structural features. An initial proof-of-principle set of smFRET results demonstrates the strengths of the labeling method. Our results highlight a powerful tool for structural studies of p97 and other large protein machines.

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Structural Dynamics of the Glycine-binding Domain of the N-Methyl-d-Aspartate Receptor

Cited by 37 publications

References 31 publications

Conformational Transitions in the Glycine-Bound GluN1 NMDA Receptor LBD via Single-Molecule FRET

Conformational Transitions in the Glycine-Bound GluN1 NMDA Receptor LBD via Single-Molecule FRET

Molecular lock regulates binding of glycine to a primitive NMDA receptor

Dual Unnatural Amino Acid Incorporation and Click‐Chemistry Labeling to Enable Single‐Molecule FRET Studies of p97 Folding

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