Conformational spread and dynamics in allostery of NMDA receptors (original) (raw)
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Conformational Transitions in the Glycine-Bound GluN1 NMDA Receptor LBD via Single-Molecule FRET
Biophysical journal, 2015
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 identi...
Single-Molecule Imaging of Conformational Dynamics in a Neurotransmitter Transporter Homolog
2016
Neurotransmitter:sodium symporter (NSS) proteins, the targets of antidepressants and psychostimulants, clear neurotransmitters from the synaptic cleft in a Na +-coupled transport mechanism. Transport is thought to occur via conformational rearrangements that alternately expose the substrate-binding site to each side of the membrane, but little is known about the mechanism by which ligand binding coordinates motions at the two faces. In this dissertation, single-molecule fluorescence resonance energy transfer (smFRET) techniques are used to image the dynamics of the prokaryotic NSS LeuT with sufficient resolution to describe the conformational states at both the intra-and extracellular faces for the first time. We found that the two sides do not move as a rigid body, contrary to popular models, and that previously undetected intermediate states are associated with transport activity. We also describe how ions and substrates influence conformational dynamics to create a productive transport cycle. i TABLE OF CONTENTS List of Graphs, Images and Illustrations .
Direct Detection of Structurally Resolved Dynamics in a Multiconformation Receptor−Ligand Complex
Journal of the American Chemical Society, 2011
Structure-based drug design relies on static protein structures despite significant evidence for the need to include protein dynamics as a serious consideration. In practice, dynamic motions are neglected because they are not understood well enough to model -a situation resulting from a lack of explicit experimental examples of dynamic receptor-ligand complexes. Here, we report highresolution details of pronounced ~1 ms timescale motions of a receptor-small molecule complex using a combination of NMR and X-ray crystallography. Large conformational dynamics in Escherichia coli dihydrofolate reductase are driven by internal switching motions of the drug-like, nanomolar-affinity inhibitor. Carr-Purcell-Meiboom-Gill relaxation dispersion experiments and NOEs revealed the crystal structure to contain critical elements of the high energy protein-ligand conformation. The availability of accurate, structurally resolved dynamics in a protein-ligand complex should serve as a valuable benchmark for modeling dynamics in other receptor-ligand complexes and prediction of binding affinities.
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, 2015
Conformational dynamics plays a critical role in the activation, deactivation, and open−close activities of ion channels in living cells. Such conformational dynamics is often inhomogeneous and extremely difficult to be directly characterized by ensemble-averaged spectroscopic imaging or only by single channel patch-clamp electric recording methods. We have developed a new and combined technical approach, single-molecule patch-clamp FRET microscopy, to probe ion channel conformational dynamics in living cell by simultaneous and correlated measurements of real-time single-molecule FRET spectroscopic imaging with single-channel electric current recording. Our approach is particularly capable of resolving ion channel conformational change rate process when the channel is at its electrically off states and before the ion channel is activated, the so-called "silent time" when the electric current signals are at zero or background. We have probed NMDA (Nmethyl-D-aspartate) receptor ion channel in live HEK-293 cell, especially, the single ion channel open−close activity and its associated protein conformational changes simultaneously. Furthermore, we have revealed that the seemingly identical electrically off states are associated with multiple conformational states. On the basis of our experimental results, we have proposed a multistate clamshell model to interpret the NMDA receptor open−close dynamics.
The structure-energy landscape of NMDA receptor gating
Nature chemical biology, 2017
N-Methyl-D-aspartate (NMDA) receptors are the main calcium-permeable excitatory receptors in the mammalian central nervous system. The NMDA receptor gating is complex, exhibiting multiple closed, open, and desensitized states; however, central questions regarding the conformations and energetics of the transmembrane domains as they relate to the gating states are still unanswered. Here, using single-molecule Förster resonance energy transfer (smFRET), we map the energy landscape of the first transmembrane segment of the Rattus norvegicus NMDA receptor under resting and various liganded conditions. These results show kinetically and structurally distinct changes associated with apo, agonist-bound, and inhibited receptors linked by a linear mechanism of gating at this site. Furthermore, the smFRET data suggest that allosteric inhibition by zinc occurs by an uncoupling of the agonist-induced changes at the extracellular domains from the gating motions leading to an apo-like state, whil...
Journal of Biological Chemistry, 2016
The N-methyl-D-aspartate (NMDA) receptors are heteromeric non-selective cation channels that require the binding of glycine and glutamate for gating. Based on crystal structures, the mechanism of partial agonism at the glycine-binding site is thought to be mediated by a shift in the conformational equilibrium between an open clamshell and a closed clamshell-like structure of the bilobed ligand-binding domain (LBD). Using single-molecule Förster resonance energy transfer (smFRET) and multiparameter fluorescence detection, which allows us to study the conformational states and dynamics in the submillisecond time scale, we show that there are at least three conformational states explored by the LBD: the low FRET, medium FRET, and high FRET states. The distance of the medium and low FRET states corresponds to what has been observed in crystallography structures. We show that the high FRET state, which would represent a more closed clamshell conformation than that observed in the crystal structure, is most likely the state initiating activation, as evidenced by the fact that the fraction of the protein in this state correlates well with the extent of activation. Furthermore, full agonist bound LBDs show faster dynamic motions between the medium and high FRET states, whereas they show slower dynamics when bound to weaker agonists or to antagonists. * This work was supported by National Institutes of Health Grants R01 GM094246 (to V. J.) and NIGMS Grant GM089657 (to D. M. D.), start-up funds from Clemson University (to H. S.), and a Schissler Foundation Fellowship for Translational Studies of Common Human Diseases (to D. M. D.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors declare that they have no conflicts of interest with the contents of this article.
The Journal of Chemical Physics, 2015
The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The coexistence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.
2020
The complex pharmacology of G-protein-coupled receptors (GPCRs) is defined by their multi-state conformational dynamics. Single-molecule Förster Resonance Energy Transfer (smFRET) is well-suited to quantify dynamics for individual protein molecules, however, its application to GPCRs is challenging; therefore, smFRET has been limited to studies of interreceptor interactions in cellular membranes and receptors in detergent environments. Here, we performed smFRET experiments on functionally active human A2A adenosine receptor (A2AAR) molecules embedded in freely diffusing lipid nanodiscs to study their intramolecular conformational dynamics. We propose a dynamic model of A2AAR activation that involves a slow (>2 ms) exchange between the active-like and inactive-like conformations in both apo and antagonist-bound A2AAR, explaining the receptor’s constitutive activity. For the agonist-bound A2AAR, we detected faster (390±80 μs) ligand efficacy-dependent dynamics. This work establishes...