Dataset Information

Slow conformational dynamics of the human A_2A adenosine receptor are temporally ordered.

ABSTRACT: A more complete depiction of protein energy landscapes includes the identification of different function-related conformational states and the determination of the pathways connecting them. We used total internal reflection fluorescence (TIRF) imaging to investigate the conformational dynamics of the human A_2A adenosine receptor (A_2AAR), a class A G protein-coupled receptor (GPCR), at the single-molecule level. Slow, reversible conformational exchange was observed among three different fluorescence emission states populated for agonist-bound A_2AAR. Transitions among these states predominantly occurred in a specific order, and exchange between inactive and active-like conformations proceeded through an intermediate state. Models derived from molecular dynamics simulations with available A_2AAR structures rationalized the relative fluorescence emission intensities for the highest and lowest emission states but not the transition state. This suggests that the functionally critical intermediate state required to achieve activation is not currently visualized among available A_2AAR structures.

SUBMITTER: Wei S

PROVIDER: S-EPMC8897252 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Slow conformational dynamics of the human A2A adenosine receptor are temporally ordered.

Wei Shushu S Thakur Naveen N Ray Arka P AP Jin Beining B Obeng Samuel S McCurdy Christopher R CR McMahon Lance R LR Gutiérrez-de-Terán Hugo H Eddy Matthew T MT Lamichhane Rajan R

Structure (London, England : 1993) 20211210 3

A more complete depiction of protein energy landscapes includes the identification of different function-related conformational states and the determination of the pathways connecting them. We used total internal reflection fluorescence (TIRF) imaging to investigate the conformational dynamics of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR), at the single-molecule level. Slow, reversible conformational exchange was observed among thr ...[more]

PMID: 34895472

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Project description: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. 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.

Dataset Information

Slow conformational dynamics of the human A_2A adenosine receptor are temporally ordered.

Publications

Slow conformational dynamics of the human A<sub>2A</sub> adenosine receptor are temporally ordered.

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