Ligand-induced conformational changes in a SMALP-encapsulated GPCR

Area of Science:

• Biochemistry
• Structural Biology
• Pharmacology

Background:

• The adenosine 2A receptor (A2AR), a G-protein-coupled receptor (GPCR), plays a crucial role in various physiological processes.
• Studying GPCRs in a native-like membrane environment is essential for understanding their function and drug interactions.

Purpose of the Study:

• To solubilize and purify the A2AR in styrene maleic acid lipid particles (SMALPs) to maintain its native membrane environment.
• To investigate the conformational changes of the A2AR upon binding of an inverse agonist (ZM241385) and an agonist (NECA) using fluorescence spectroscopy.

Main Methods:

• Solubilization and purification of A2AR using SMALPs.
• Characterization of A2AR-SMALPs using fluorescence emission spectroscopy.
• Site-specific labeling with IAEDANS to probe conformational dynamics.
• Mutagenesis studies involving tryptophan to tyrosine substitutions.

Main Results:

• A2AR-SMALPs retained native phospholipids from Pichia pastoris, confirming a native-like environment.
• The inverse agonist ZM241385 induced significant changes in A2AR fluorescence, including increased emission and a red-shift, suggesting altered tryptophan environments and increased hydrophobicity.
• ZM241385 binding led to a more hydrophobic environment at the intracellular end of TM6, consistent with G-protein binding crevice closure.
• The agonist NECA showed only minor effects on A2AR fluorescence compared to ZM241385.

Conclusions:

• SMALP encapsulation successfully preserved the native environment and constitutive activity of the A2AR.
• ZM241385 binding induces distinct conformational transitions in the A2AR, impacting tryptophan residue environments and receptor dynamics.
• The A2AR exhibits differential responses to inverse agonists and agonists, with NECA showing limited conformational effects in this system.