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Mapping Interaction Sites on Human Chemokine Receptors by Deep Mutational Scanning
Jeremiah D Heredia1, Jihye Park1, Riley J Brubaker1
1Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
Abstract:
Chemokine receptors CXCR4 and CCR5 regulate WBC trafficking and are engaged by the HIV-1 envelope glycoprotein gp120 during infection. We combine a selection of human CXCR4 and CCR5 libraries comprising nearly all of ∼7000 single amino acid substitutions with deep sequencing to define sequence-activity landscapes for surface expression and ligand interactions. After consideration of sequence constraints for surface expression, known interaction sites with HIV-1-blocking Abs were appropriately identified as conserved residues following library sorting for Ab binding, validating the use of deep mutational scanning to map functional interaction sites in G protein-coupled receptors. Chemokine CXCL12 was found to interact with residues extending asymmetrically into the CXCR4 ligand-binding cavity, similar to the binding surface of CXCR4 recognized by an antagonistic viral chemokine previously observed crystallographically. CXCR4 mutations distal from the chemokine binding site were identified that enhance chemokine recognition. This included disruptive mutations in the G protein-coupling site that diminished calcium mobilization, as well as conservative mutations to a membrane-exposed site (CXCR4 residues H792.45 and W1614.50) that increased ligand binding without loss of signaling. Compared with CXCR4-CXCL12 interactions, CCR5 residues conserved for gp120 (HIV-1 BaL strain) interactions map to a more expansive surface, mimicking how the cognate chemokine CCL5 makes contacts across the entire CCR5 binding cavity. Acidic substitutions in the CCR5 N terminus and extracellular loops enhanced gp120 binding. This study demonstrates how comprehensive mutational scanning can define functional interaction sites on receptors, and novel mutations that enhance receptor activities can be found simultaneously.
Insights
This study maps functional sites on chemokine receptors CXCR4 and CCR5 using deep mutational scanning. Novel mutations were found that enhance receptor activity and ligand binding, offering new insights into HIV-1 entry and WBC trafficking.
Area of Science:
- Molecular Biology
- Immunology
- Virology
Background:
- Chemokine receptors CXCR4 and CCR5 are crucial for white blood cell (WBC) trafficking and serve as entry points for HIV-1 infection via the envelope glycoprotein gp120.
- Understanding the precise interactions between these receptors and their ligands is vital for developing therapeutic strategies against viral infections and inflammatory diseases.
Purpose of the Study:
- To comprehensively map the sequence-activity landscapes of CXCR4 and CCR5, defining residues critical for surface expression, ligand binding, and G protein signaling.
- To identify novel mutations that enhance receptor activity and ligand interactions, particularly in the context of HIV-1 binding.
Main Methods:
- Utilized deep mutational scanning of large, comprehensive human CXCR4 and CCR5 single amino acid substitution libraries (∼7000 substitutions each).
- Employed deep sequencing to analyze library sorting results for surface expression, antibody binding (HIV-1 blocking), and chemokine interactions (CXCL12 for CXCR4, gp120 for CCR5).
- Correlated mutational effects with known functional sites, including G protein-coupling domains and ligand-binding pockets.
Main Results:
- Deep mutational scanning successfully identified conserved residues at known interaction sites, validating the method for mapping functional hotspots in G protein-coupled receptors.
- CXCR4 interactions with CXCL12 involve residues extending into the binding cavity, with distal mutations found to enhance chemokine recognition and ligand binding without compromising signaling.
- CCR5 interactions with HIV-1 gp120 involve a broader surface compared to CXCR4-CXCL12, with acidic substitutions in the N-terminus and extracellular loops enhancing gp120 binding.
Conclusions:
- Comprehensive mutational scanning is a powerful approach to define functional interaction sites on chemokine receptors.
- Novel mutations enhancing CXCR4 and CCR5 activity and ligand binding were identified, providing potential targets for therapeutic intervention.
- The study elucidates distinct interaction mechanisms for CXCR4 and CCR5 with their respective ligands and HIV-1, informing future drug design.
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