High throughput mutational characterization of the GPCR ligand C5a using yeast display and deep sequencing

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• High-throughput mutagenesis is crucial for characterizing protein functions and drug development.
• G protein-coupled receptors (GPCRs) are a major target class, but ligand mutagenesis studies are challenging due to receptor solubilization difficulties.
• Understanding GPCR-ligand interactions is vital for therapeutic advancements.

Purpose of the Study:

• To develop a novel platform for stabilizing membrane receptors in lipid vesicles for direct ligand screening.
• To investigate the impact of mutations on the binding of anaphylatoxin complement 5a (C5a) to its receptors, C5aR1 and C5aR2.
• To identify novel ligands with selective activity towards C5aR2.

Main Methods:

• Development of a lipid vesicle-based platform for embedding and stabilizing membrane receptors.
• Application of the platform for high-throughput screening of mutations affecting C5a binding to C5aR1 and C5aR2.
• Analysis of structure-activity relationships to understand C5a-GPCR interactions.

Main Results:

• The lipid vesicle platform successfully stabilized membrane receptors, enabling direct ligand screening.
• Mutagenesis studies provided new insights into the molecular basis of C5a interaction with C5aR1 and C5aR2.
• Discovery of novel ligands demonstrating selective activation of C5aR2 over C5aR1.

Conclusions:

• The lipid vesicle-based approach overcomes previous limitations in studying GPCR-ligand interactions.
• The findings deepen the understanding of C5a receptor pharmacology.
• The identified selective C5aR2 ligands represent potential therapeutic leads for inflammatory diseases.