Antibody epitopes on g protein-coupled receptors mapped with genetically encoded photoactivatable cross-linkers.
We developed a strategy for creating epitope maps of monoclonal antibodies (mAbs) that bind to G protein-coupled receptors (GPCRs) containing photo-cross-linkers. Using human CXC chemokine receptor 4 (CXCR4) as a model system, we genetically incorporated the photolabile unnatural amino acid p-azido-l-phenylalanine (azF) at various positions within extracellular loop 2 (EC2). We then mapped the interactions of the azF-CXCR4 variants with mAb 12G5 using targeted loss-of-function studies and photo-cross-linking in whole cells in a microplate-based format. We used a novel variation of a whole cell enzyme-linked immunosorbent assay to quantitate cross-linking efficiency. 12G5 cross-linked primarily to residues 184, 178, and 189 in EC2 of CXCR4. Mapping of the data to the crystal structure of CXCR4 showed a distinct mAb epitope footprint with the photo-cross-linked residues clustered around the loss-of-function sites. We also used the targeted photo-cross-linking approach to study the interaction of human CC chemokine receptor 5 (CCR5) with PRO 140, a humanized mAb that inhibits human immunodeficiency virus-1 cellular entry, and 2D7. The mAbs produced distinct cross-linking patterns on EC2 of CCR5. PRO 140 cross-linked primarily to residues 174 and 175 at the amino-terminal end of EC2, and 2D7 cross-linked mainly to residues 170, 176, and 184. These results were mapped to the recent crystal structure of CCR5 in complex with maraviroc, showing cross-linked residues at the tip of the maraviroc binding crevice formed by EC2. As a strategy for mapping mAb epitopes on GPCRs, our targeted photo-cross-linking method is complementary to loss-of-function mutagenesis results and should be especially useful for studying mAbs with discontinuous epitopes.
Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University , 1230 York Avenue, New York, New York 10065, United States.
Sarmistha
S
Ray-Saha
Thomas
T
Huber
Thomas P
TP
Sakmar
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001386:Azides / Q000737:chemistry*; D018604:Epitope Mapping / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D055668:Photochemical Processes; D011487:Protein Conformation; D015202:Protein Engineering; D019713:Receptors, CCR5 / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D019718:Receptors, CXCR4 / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*
D000911:Antibodies, Monoclonal; D001386:Azides; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D043562:Receptors, G-Protein-Coupled; C027162:4-azidophenylalanine; D010649:Phenylalanine