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

• Published in: Structure (London) 2022-03, Vol.30 (3), p.329-337.e5
• Main Authors: Wei, Shushu, Thakur, Naveen, Ray, Arka P., Jin, Beining, Obeng, Samuel, McCurdy, Christopher R., McMahon, Lance R., Gutiérrez-de-Terán, Hugo, Eddy, Matthew T., Lamichhane, Rajan
• Format: Article
• Language: English
• Subjects: A2A adenosine receptor; biophysics; conformational dynamics; G-protein-coupled receptors; lipid nanodiscs; membrane proteins; single-molecule fluorescence spectroscopy; TIRF
• ISSN: 0969-2126; 1878-4186; 1878-4186
• DOI: 10.1016/j.str.2021.11.005

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 three different fluorescence emission states populated for agonist-bound A2AAR. 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 A2AAR 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 A2AAR structures. [Display omitted] •Single-molecule fluorescence observes slow dynamic behavior of A2AAR complexes•Three different fluorescence emission states observed for A2AAR complexes with agonists•Reversible transitions among different states occurred in a sequential order•A functionally critical transition state not represented among available structures Wei et al. use single-molecule fluorescence to observe slow exchange among at least three conformations of the agonist-bound A2A adenosine receptor. Transitions from inactive to active-like conformations are sequential and must proceed through an intermediate state not currently represented among available structures.