High throughput mutational characterization of the GPCR ligand C5a using yeast display and deep sequencing
Description
High-throughput mutagenesis platforms are powerful tools to systematically characterize protein functions, and play a crutial role in therapeutic developments. As the largest class of membrane receptors and drug targets, G protein-coupled receptors (GPCRs) have been the focus of these studies. While significant progress has been made in characterizing the receptors, mutagenesis studies on their ligands have lagged behind, because of the challenges in solubilizing the target receptor. In the publication titled "High throughput mutational characterization of the GPCR ligand C5a using yeast display and deep sequencing", we present a novel approach that utilizes lipid vesicles to embed and stabilize target membrane receptors, allowing direct ligand screening. Using this platform, we investigate the anaphylatoxin Complement 5a (C5a) and characterized the mutational impacts on its interactions with the two native GPCRs: complement 5a receptor 1 (C5aR1) and complement 5a receptor 2 (C5L2). The screening provided new insights into the molecular basis of the interaction with the two receptors, and led to the discovery of novel ligands that selectively activate C5L2, but not C5aR1. This highlights the potential of our method to advance our understanding of GPCR ligands and paves the way for designing novel ligands with therapeutically values for this important class of receptors. The dataset includes the sequencing data and the relevant python code to reproduce the results presented in the manuscript. The sequencing data includes the raw FASTQ files and processed FASTA files obtained from the mutagenesis analysis of C5a binding to its receptors, C5aR1 and C5L2. The FASTQ files are stored under folder "FASTAQ_SSM-C5a_MutagenesisSorting", and the FASTA files are stored under "fasta_files".
Files
Steps to reproduce
The dataset includes sequencing data that was collected from a high-throughput mutagenesis experiment analyzing the binding properties of C5a to its receptors, C5aR1 and C5L2. Briefly, a site-saturation mutagenesis library of C5a was expressed on the yeast surface, and binding assays were conducted using fluorescently labeled C5aR1 and C5L2. Cells were sorted into populations based on fluorescence intensity via flow cytometry, and the abundance of individual mutants in the sorted population is determined using deep sequencing. The enrichment of individual mutants within these sorted populations reflects their binding affinities to the targets, and can provide valuable information on how specific mutations on C5a affects its binding to the target receptors. To reproduce the results in the publication, please run code: main_reproduce_C5aR1.py and main_reproduce_C5L2.py in the Python code folder.
Institutions
- Stanford UniversityCA, Stanford