Structure and function of an irreversible agonist-β(2) adrenoceptor complex.

Area of Science:

• Structural biology
• Biochemistry
• Molecular pharmacology

Background:

• G-protein-coupled receptors (GPCRs) are crucial cell signaling proteins, but agonist binding mechanisms remain unclear.
• Understanding GPCR activation is key for drug design, yet crystallizing agonist-bound states is challenging due to weak ligand interactions.
• The human beta-2 adrenergic receptor (β(2)AR) is a well-studied GPCR, but its active conformation is difficult to capture structurally.

Purpose of the Study:

• To determine the high-resolution structure of an agonist-bound GPCR.
• To elucidate the molecular basis of GPCR activation and allosteric modulation.
• To enable structure-guided design of novel GPCR-targeting therapeutics.

Main Methods:

• Design and synthesis of a novel covalent agonist for the β(2)AR, tethered via a disulfide bond.
• Formation of a stable covalent β(2)AR-agonist complex capable of G protein activation.
• Crystallization of the covalent agonist-bound β(2)AR-T4L fusion protein in lipid bilayers using the lipidic mesophase method.
• Determination of the complex's structure at 3.5 Å resolution.

Main Results:

• A stable covalent agonist-bound β(2)AR complex was successfully created and crystallized.
• Structural analysis revealed the receptor's active conformation, requiring interactions at both extracellular and intracellular surfaces.
• Molecular dynamics simulations (up to 30 µs) showed the active state's instability without G protein or stabilizing antibody.

Conclusions:

• A covalent agonist strategy overcomes challenges in crystallizing agonist-bound GPCRs.
• The determined structure provides critical insights into the conformational changes required for β(2)AR activation.
• This work advances the understanding of GPCR activation mechanisms and facilitates structure-based drug discovery.