scholarly journals Conformational dynamics between transmembrane domains and allosteric modulation of a metabotropic glutamate receptor

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Vanessa A Gutzeit ◽  
Jordana Thibado ◽  
Daniel Starer Stor ◽  
Zhou Zhou ◽  
Scott C Blanchard ◽  
...  
Keyword(s):  
Single Molecule ◽  
Metabotropic Glutamate Receptors ◽  
Metabotropic Glutamate Receptor ◽  
Conformational Dynamics ◽  
Resonance Energy ◽  
Transmembrane Domains ◽  
Metabotropic Glutamate ◽  
Receptor Modulation ◽  
Gpcr Activation ◽  
Class C

Metabotropic glutamate receptors (mGluRs) are class C, synaptic G-protein-coupled receptors (GPCRs) that contain large extracellular ligand binding domains (LBDs) and form constitutive dimers. Despite the existence of a detailed picture of inter-LBD conformational dynamics and structural snapshots of both isolated domains and full-length receptors, it remains unclear how mGluR activation proceeds at the level of the transmembrane domains (TMDs) and how TMD-targeting allosteric drugs exert their effects. Here, we use time-resolved functional and conformational assays to dissect the mechanisms by which allosteric drugs activate and modulate mGluR2. Single-molecule subunit counting and inter-TMD fluorescence resonance energy transfer measurements in living cells reveal LBD-independent conformational rearrangements between TMD dimers during receptor modulation. Using these assays along with functional readouts, we uncover heterogeneity in the magnitude, direction, and the timing of the action of both positive and negative allosteric drugs. Together our experiments lead to a three-state model of TMD activation, which provides a framework for understanding how inter-subunit rearrangements drive class C GPCR activation.

scholarly journals Single molecule analysis reveals positive allosteric modulators are required to stabilize the active state of a metabotropic glutamate receptor

2021 ◽  
Author(s):  
Anne-Marinette Cao ◽  
Robert B. Quast ◽  
Fataneh Fatemi ◽  
Philippe Rondard ◽  
Jean-Philippe Pin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Metabotropic Glutamate Receptors ◽  
Metabotropic Glutamate Receptor ◽  
Brain Diseases ◽  
Allosteric Modulators ◽  
Fast Kinetics ◽  
Single Molecule Level ◽  
Metabotropic Glutamate ◽  
Subtype Selectivity ◽  
Positive Allosteric Modulators

AbstractMetabotropic glutamate receptors (mGlu) are G protein-coupled receptors that represent promising targets for brain diseases. Much hope in drug development come from the discovery of positive allosteric modulators that display subtype selectivity, and act by increasing agonist potency, as well as efficacy in most cases. How such compounds can influence agonist efficacy remains unclear. Here, we explore the structural dynamics of the full-length mGlu2 dimers at submillisecond timescales using single molecule FRET on diffusing receptors in optimized detergent micelles. We show that glutamate binding in the Venus flytrap extracellular domains does not stabilize fully the receptors in their active states. The full activation of all receptors can only be observed in the presence of either a positive allosteric modulator or the Gi protein. Our results provide important new insights on the fast kinetics and the action of the allosteric modulators on mGlu activation at the single molecule level.

scholarly journals Structure of a Class C GPCR Metabotropic Glutamate Receptor 1 Bound to an Allosteric Modulator

Science ◽  
2014 ◽  
Vol 344 (6179) ◽  
pp. 58-64 ◽  
Author(s):  
Huixian Wu ◽  
Chong Wang ◽  
Karen J. Gregory ◽  
Gye Won Han ◽  
Hyekyung P. Cho ◽  
...  
Keyword(s):  
Metabotropic Glutamate Receptors ◽  
Metabotropic Glutamate Receptor ◽  
Full Length ◽  
Activation Mechanism ◽  
Allosteric Modulator ◽  
Excitatory Neurotransmitter ◽  
Metabotropic Glutamate ◽  
Metabotropic Glutamate Receptor 1 ◽  
Negative Allosteric Modulator ◽  
Class C

The excitatory neurotransmitter glutamate induces modulatory actions via the metabotropic glutamate receptors (mGlus), which are class C G protein–coupled receptors (GPCRs). We determined the structure of the human mGlu1 receptor seven-transmembrane (7TM) domain bound to a negative allosteric modulator, FITM, at a resolution of 2.8 angstroms. The modulator binding site partially overlaps with the orthosteric binding sites of class A GPCRs but is more restricted than most other GPCRs. We observed a parallel 7TM dimer mediated by cholesterols, which suggests that signaling initiated by glutamate’s interaction with the extracellular domain might be mediated via 7TM interactions within the full-length receptor dimer. A combination of crystallography, structure-activity relationships, mutagenesis, and full-length dimer modeling provides insights about the allosteric modulation and activation mechanism of class C GPCRs.

Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

2018 ◽  
Author(s):  
Alexander Carl DeHaven
Keyword(s):  
Energy Transfer ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
U2 Snrna ◽  
Folding Dynamics ◽  
The Impact

This thesis contains four topic areas: a review of single-molecule microscropy methods and splicing, conformational dynamics of stem II of the U2 snRNA, the impact of post-transcriptional modifications on U2 snRNA folding dynamics, and preliminary findings on Mango aptamer folding dynamics.

scholarly journals Structural Characterization of Receptor–Receptor Interactions in the Allosteric Modulation of G Protein-Coupled Receptor (GPCR) Dimers

2021 ◽  
Vol 22 (6) ◽  
pp. 3241
Author(s):  
Raudah Lazim ◽  
Donghyuk Suh ◽  
Jai Woo Lee ◽  
Thi Ngoc Lan Vu ◽  
Sanghee Yoon ◽  
...  
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Metabotropic Glutamate Receptor ◽  
Three Dimensional ◽  
Allosteric Modulation ◽  
Experimental Investigations ◽  
G Protein Coupled Receptor ◽  
Metabotropic Glutamate ◽  
Gpcr Activation ◽  
G Protein Coupled

G protein-coupled receptor (GPCR) oligomerization, while contentious, continues to attract the attention of researchers. Numerous experimental investigations have validated the presence of GPCR dimers, and the relevance of dimerization in the effectuation of physiological functions intensifies the attractiveness of this concept as a potential therapeutic target. GPCRs, as a single entity, have been the main source of scrutiny for drug design objectives for multiple diseases such as cancer, inflammation, cardiac, and respiratory diseases. The existence of dimers broadens the research scope of GPCR functions, revealing new signaling pathways that can be targeted for disease pathogenesis that have not previously been reported when GPCRs were only viewed in their monomeric form. This review will highlight several aspects of GPCR dimerization, which include a summary of the structural elucidation of the allosteric modulation of class C GPCR activation offered through recent solutions to the three-dimensional, full-length structures of metabotropic glutamate receptor and γ-aminobutyric acid B receptor as well as the role of dimerization in the modification of GPCR function and allostery. With the growing influence of computational methods in the study of GPCRs, we will also be reviewing recent computational tools that have been utilized to map protein–protein interactions (PPI).

scholarly journals Total workflows of the single-molecule imaging analysis in living cells: a tutorial guidance to the measurement of the drug effects on a GPCR

2020 ◽  
Author(s):  
Masataka Yanagawa ◽  
Yasushi Sako
Keyword(s):  
Single Molecule ◽  
Metabotropic Glutamate Receptor ◽  
Particle Density ◽  
Drug Effects ◽  
Quantitative Information ◽  
Single Molecule Imaging ◽  
Single Molecule Tracking ◽  
Metabotropic Glutamate ◽  
Protein Protein Interaction ◽  
Diffusion Dynamics

AbstractSingle-molecule imaging (SMI) is a powerful method to measure the dynamics of membrane proteins on the cell membrane. The single-molecule tracking (SMT) analysis provides information about the diffusion dynamics, the oligomer size distribution, and the particle density change. The affinity and on/off-rate of a protein—protein interaction can be estimated from the dual-color SMI analysis. However, it is difficult for trainees to determine quantitative information from the SMI movies. The present protocol guides the detailed workflows to measure the drug-activated dynamics of a G protein-coupled receptor (GPCR) and metabotropic glutamate receptor 3 (mGluR3), by using the total internal reflection fluorescence microscopy (TIRFM). This tutorial guidance comprises an open-source software named smDynamicsAnalyzer, with which one can easily analyze the SMT dataset by just following the workflows after building a designated folder structure (https://github.com/masataka-yanagawa/IgorPro8-smDynamicsAnalyzer).

scholarly journals Allosteric modulation of the SARS-CoV-2 spike conformation

2021 ◽  
Author(s):  
Marco A Diaz-Salinas ◽  
Qi Li ◽  
Monir Ejemel ◽  
Yang Wang ◽  
James B Munro
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Data ◽  
Correlation Spectroscopy ◽  
Host Cells ◽  
Parallel Solution ◽  
Real Time Analysis ◽  
Multiple Conformations

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through binding to angiotensin-converting enzyme 2 (ACE2), which is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural data and real-time analysis of conformational dynamics have shown that S can adopt multiple conformations, which mediate the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We found that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicated antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

scholarly journals Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling

2018 ◽  
Author(s):  
Robert B. Quast ◽  
Fataneh Fatemi ◽  
Michel Kranendonk ◽  
Emmanuel Margeat ◽  
Gilles Truan
Keyword(s):  
Single Molecule ◽  
Conformational Equilibrium ◽  
Fluorescent Dyes ◽  
Photophysical Properties ◽  
Accurate Determination ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
General Strategy

ABSTRACTConjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Förster resonance energy transfer (smFRET) - can lead to substantial changes of the dye’s photophysical properties, ultimately biasing the quantitative determination of inter-dye distances. In particular the popular cyanine dyes and their derivatives, which are by far the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli was developed. Applied to human NADPH- cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated. Furthermore, the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.

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