Skip to main content
OSTI.GOVU.S. Department of Energy Office of Scientific and Technical Information
U.S. Department of Energy
Office of Scientific and Technical Information
 

Ligand modulation of the conformational dynamics of the A2A adenosine receptor revealed by single-molecule fluorescence

Journal Article · · Scientific Reports
 [1];  [2];  [1];  [1];  [3];  [4];  [4];  [5];  [6];  [7];  [1]
  1. Univ. of Toronto, ON (Canada); Univ. of Toronto Mississauga, ON (Canada)
  2. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
  3. Univ. of Toronto Mississauga, ON (Canada)
  4. Univ. of Toronto Mississauga, ON (Canada); Univ. of Toronto, ON (Canada)
  5. Univ. of South Florida, Tampa, FL (United States)
  6. Univ. of Toronto Mississauga, ON (Canada); Univ. of South Florida, Tampa, FL (United States); Moffitt Cancer Center, Tampa, FL (United States)
  7. Univ. of Toronto Mississauga, ON (Canada) Univ. of Toronto, ON (Canada)
+ Show Author Affiliations
G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 2296.31 on TM6 was measured using fluorescence correlation spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150–350 ns), intermediate (50–60 μs) and slow (200–300 μs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y2887.53, and Y2907.55) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 2296.31 on TM6. As a result, this study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.
Research Organization:
Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
Sponsoring Organization:
Canadian Inst. for Health Research; LDRD; Natural Sciences and Engineering Research Council of Canada; USDOE National Nuclear Security Administration (NNSA)
Grant/Contract Number:
89233218CNA000001; AC52-06NA25396
OSTI ID:
1804394
Report Number(s):
LA-UR--20-23955
Journal Information:
Scientific Reports, Journal Name: Scientific Reports Journal Issue: 1 Vol. 11; ISSN 2045-2322
Publisher:
Nature Publishing GroupCopyright Statement
Country of Publication:
United States
Language:
English

References (59)

A Close Look at Fluorescence Quenching of Organic Dyes by Tryptophan journal November 2005
Fluorescence Quenching by Photoinduced Electron Transfer: A Reporter for Conformational Dynamics of Macromolecules journal July 2009
Conformational selection in protein binding and function: Conformational Selection journal September 2014
A comparison of chemical shift sensitivity of trifluoromethyl tags: optimizing resolution in 19F NMR studies of proteins journal March 2015
VMD: Visual molecular dynamics journal February 1996
Insights into the conformations and dynamics of intrinsically disordered proteins using single-molecule fluorescence journal November 2017
Concerted Interconversion between Ionic Lock Substates of the β2 Adrenergic Receptor Revealed by Microsecond Timescale Molecular Dynamics journal January 2010
Can Specific Protein-Lipid Interactions Stabilize an Active State of the Beta 2 Adrenergic Receptor? journal October 2015
Ligand-Induced Coupling between Oligomers of the M2 Receptor and the Gi1 Protein in Live Cells journal September 2018
The Dynamic Process of β2-Adrenergic Receptor Activation journal January 2013
Structural Insights into the Dynamic Process of β 2 -Adrenergic Receptor Signaling journal May 2015
Angiotensin Analogs with Divergent Bias Stabilize Distinct Receptor Conformations journal January 2019
Studying GPCR conformational dynamics by single molecule fluorescence journal August 2019
Structure of the Adenosine A2A Receptor in Complex with ZM241385 and the Xanthines XAC and Caffeine journal September 2011
Qualitative analysis by online nuclear magnetic resonance using Carr–Purcell–Meiboom–Gill sequence with low refocusing flip angles journal March 2011
A G protein-coupled receptor at work: the rhodopsin model journal November 2009
Structural Basis for G Protein-Coupled Receptor Activation journal September 2017
Labeling and Single-Molecule Methods To Monitor G Protein-Coupled Receptor Dynamics journal June 2016
Inter- and Intramolecular Fluorescence Quenching of Organic Dyes by Tryptophan journal November 2003
Microbial and Animal Rhodopsins: Structures, Functions, and Molecular Mechanisms journal September 2013
Adenosine A 2A Receptor as a Drug Discovery Target journal November 2013
FRET Efficiency Distributions of Multistate Single Molecules journal November 2010
Sub-Diffusion Decays in Fluorescence Correlation Spectroscopy: Dye Photophysics or Protein Dynamics? journal April 2013
The Effect of Intrachain Electrostatic Repulsion on Conformational Disorder and Dynamics of the Sic1 Protein journal April 2014
Photoinduced Electron Transfer and Fluorophore Motion as a Probe of the Conformational Dynamics of Membrane Proteins: Application to the Influenza A M2 Proton Channel journal April 2011
Crystal structure of metarhodopsin II journal March 2011
Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation journal May 2011
Crystal structure of the β2 adrenergic receptor–Gs protein complex journal July 2011
Conformational dynamics of a class C G-protein-coupled receptor journal August 2015
Activation of the A2A adenosine G-protein-coupled receptor by conformational selection journal May 2016
Structure of the adenosine A2A receptor bound to an engineered G protein journal July 2016
Single-molecule analysis of ligand efficacy in β2AR–G-protein activation journal June 2017
Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy journal October 2014
A photoprotection strategy for microsecond-resolution single-molecule fluorescence spectroscopy journal January 2011
How many drug targets are there? journal December 2006
Structural absorption by barbule microstructures of super black bird of paradise feathers journal January 2018
Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study journal August 2018
Ror2 signaling regulates Golgi structure and transport through IFT20 for tumor invasiveness journal January 2017
Electronic ground and excited state properties of dipyrrometheneboron difluoride (BODIPY): Dimers with application to biosciences journal January 2002
Characterizing rhodopsin signaling by EPR spectroscopy: from structure to dynamics journal January 2015
Fluorescent PET (Photoinduced Electron Transfer) sensors as potent analytical tools journal January 2009
Fluorescence anisotropy: from single molecules to live cells journal January 2010
Direct observation of ultrafast folding and denatured state dynamics in single protein molecules journal October 2009
Integrated view of internal friction in unfolded proteins from single-molecule FRET, contact quenching, theory, and simulations journal February 2017
G i - and G s -coupled GPCRs show different modes of G-protein binding journal February 2018
A 2A adenosine receptor functional states characterized by 19 F-NMR journal November 2018
LigParGen web server: an automatic OPLS-AA parameter generator for organic ligands journal April 2017
A new look at the statistical model identification journal December 1974
Pharmacology and Structure of Isolated Conformations of the Adenosine A 2A Receptor Define Ligand Efficacy journal February 2013
The G-Protein-Coupled Receptors in the Human Genome Form Five Main Families. Phylogenetic Analysis, Paralogon Groups, and Fingerprints journal June 2003
The 2.6 Angstrom Crystal Structure of a Human A2A Adenosine Receptor Bound to an Antagonist journal November 2008
Structure of an Agonist-Bound Human A2A Adenosine Receptor journal March 2011
Structural Basis for Allosteric Regulation of GPCRs by Sodium Ions journal July 2012
The Molecular Basis of G Protein–Coupled Receptor Activation journal June 2018
Cryo-Electron Microscopy: Moving Beyond X-Ray Crystal Structures for Drug Receptors and Drug Development journal January 2020
An Introduction to the Bootstrap book May 1994
Accurate FRET Measurements within Single Diffusing Biomolecules Using Alternating-Laser Excitation journal April 2005
Bodipy Derivatives as Triplet Photosensitizers and the Related Intersystem Crossing Mechanisms journal December 2019
Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein journal May 2018

Similar Records

Intermediate-state-trapped mutants pinpoint G protein-coupled receptor conformational allostery
Journal Article · Thu Mar 09 19:00:00 EST 2023 · Nature Communications · OSTI ID:2469627
Ligand modulation of sidechain dynamics in a wild-type human GPCR
Journal Article · Thu Oct 05 20:00:00 EDT 2017 · eLife · OSTI ID:1628874
A functional selectivity mechanism at the serotonin-2A GPCR involves ligand-dependent conformations of intracellular loop 2
Journal Article · Mon Oct 13 20:00:00 EDT 2014 · Journal of the American Chemical Society · OSTI ID:1345813

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Biochemistry
Biological science
Biophysical chemistry
Biophysics
Computational biophysics
Molecular biology
Proteins
Single-molecule biophysics
Structural biology