Mapping Interaction Sites on Human Chemokine Receptors by Deep Mutational Scanning

Chemokine receptors CXCR4 and CCR5 regulate white blood cell trafficking, and are engaged by the HIV-1 envelope glycoprotein gp120 during infection. We combine directed evolution of CXCR4 and CCR5 libraries comprising nearly all ~7,000 single amino acid substitutions with deep sequencing to define sequence-fitness landscapes for surface expression and ligand interactions. Functional interaction sites are mapped based on conservation; for example, extracellular residues are conserved for binding HIV-1-blocking antibodies, as expected. Chemokine CXCL12 interacts with residues extending asymmetrically into the CXCR4 ligand-binding cavity, and distal mutations within allosteric and G protein coupling sites are identified that enhance chemokine binding. CCR5 residues conserved for gp120 interactions partially overlap with the chemokine-binding site, and gp120 binding is increased by acidic substitutions in the CCR5 N-terminus and extracellular loops. Furthermore, general features are apparent from sequence patterns, including membrane regions that are intolerant to polar mutations, and deleterious cysteine substitutions within extracellular loops. Single-site saturation mutagenesis libraries were constructed of human CXCR4 and CCR5, and expressed in human Expi293F cells (a HEK293 derivative). Cells were evolved by FACS for surface expression and binding to protein ligands. Frequencies of variants in the sorted population (measured from RNA transcripts) were compared to frequencies in the DNA libraries to calculate log(base2) enrichment ratios for all amino acid substitutions. All evolution experiments were in duplicate.