Production of human A2AAR in lipid nanodiscs for 19F-NMR and single-molecule fluorescence spectroscopy

Thakur, Naveen; Wei, Shushu; Ray, Arka Prabha; Lamichhane, Rajan; Eddy, Matthew T.

doi:10.1016/j.xpro.2022.101535

Cited by 13 publications

(12 citation statements)

References 15 publications

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“…We expressed and purified A 2A AR and A 2A AR[W246F] following established procedures, 36 , 57 , 58 described in detail below. Expression plasmids were linearized using PmeI and transformed by electroporation into P. pastoris BG12 cells.…”

Section: Methodsmentioning

confidence: 99%

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Global insights into the fine tuning of human A2AAR conformational dynamics in a ternary complex with an engineered G protein viewed by NMR

Ferré¹,

Anazia²,

Silva³

et al. 2022

Cell Reports

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Section: Methodsmentioning

confidence: 99%

“…Clones were then screened in 4 mL cultures for receptor expression using an anti-FLAG Western blot analysis. 58 …”

Section: Methodsmentioning

confidence: 99%

Global insights into the fine tuning of human A2AAR conformational dynamics in a ternary complex with an engineered G protein viewed by NMR

Ferré¹,

Anazia²,

Silva³

et al. 2022

Cell Reports

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“…Assembly of lipid nanodiscs containing A 2A AR followed protocols from previous studies [47][48][49] optimized to facilitate samples prepared with a wider range of lipid compositions. The scaffold protein MSP1D1 was expressed and purified similarly as described in previous studies 47,48 .…”

Section: Nanodisc Assemblymentioning

confidence: 99%

Anionic phospholipids control mechanisms of GPCR-G protein recognition

et al. 2023

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G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we explore the impact of anionic lipids on the function-related conformational equilibria of the human A2A adenosine receptor (A2AAR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids prime the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeds through a less favorable induced fit mechanism. In computational models, anionic lipids mimic interactions between a G protein and positively charged residues in A2AAR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly alters the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.

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“…Nanodisc assembly. Assembly of lipid nanodiscs containing A2AAR followed protocols from previous studies [47][48][49] optimized to facilitate samples prepared with a wider range of lipid compositions. The scaffold protein MSP1D1 was expressed and purified similarly as described in previous studies 47,48 .…”

Section: A2aar Expression Purification and 19 F-labeling Via Chemical...mentioning

confidence: 99%

Anionic Phospholipids Control Mechanisms of GPCR-G Protein Recognition

Thakur

Ray

Sharp

et al. 2023

Preprint

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G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we visualized the impact of anionic lipids on the function-related conformational equilibria of the human A2A adenosine receptor (A2AAR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids primed the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeded through a less favorable induced fit mechanism. In computational models, anionic lipids mimicked interactions between a G protein and positively charged residues in A2AAR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly altered the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.

show abstract

Production of human A2AAR in lipid nanodiscs for 19F-NMR and single-molecule fluorescence spectroscopy

Cited by 13 publications

References 15 publications

Global insights into the fine tuning of human A2AAR conformational dynamics in a ternary complex with an engineered G protein viewed by NMR

Global insights into the fine tuning of human A2AAR conformational dynamics in a ternary complex with an engineered G protein viewed by NMR

Anionic phospholipids control mechanisms of GPCR-G protein recognition

Anionic Phospholipids Control Mechanisms of GPCR-G Protein Recognition

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