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Title: 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)
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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:
USDOE National Nuclear Security Administration (NNSA); USDOE Laboratory Directed Research and Development (LDRD) Program; Natural Sciences and Engineering Research Council of Canada (NSERC); Canadian Inst. for Health Research
Grant/Contract Number:
89233218CNA000001; RGPIN 2017-06030; MOP-43998; AC52-06NA25396
OSTI ID:
1804394
Report Number(s):
LA-UR-20-23955
Journal Information:
Scientific Reports, Vol. 11, Issue 1; ISSN 2045-2322
Publisher:
Nature Publishing GroupCopyright Statement
Country of Publication:
United States
Language:
English

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