Extended Data Figure 4 : Structural analysis of hCEACAM1 and hTIM-3 protein interactions.

From: CEACAM1 regulates TIM-3-mediated tolerance and exhaustion

Extended Data Figure 4

a, Schematic diagram of single-chain construct consisting of hCEACAM1 IgV-domain (amino acids 1–107), a linker consisting of (GGGGS)4 and hTIM-3 IgV-domain (amino acids 1–105) and C-terminal hexahistidine tag. be, Surface plasmon resonance analyses of hCEACAM1–hTIM-3 single-chain interaction with GST–hTIM-3. b, Representative sensorgrams of serial dilutions of hCEACAM1–hTIM-3 single chain flowed over immobilized GST–hTIM-3 or GST alone. c, Representative sensorgrams of 600 nM hCEACAM1–hTIM-3 single-chain flowed over immobilized GST–hTIM-3 in presence of various concentrations of blocking hTIM-3 specific peptide (amino acids 58–77) or control scrambled peptide. d, Representative sensorgrams as in b in presence of various concentrations of anti-hCEACAM1 monoclonal antibody (26H7) or control isotype antibody (mIgG1, MOPC). e, Bar graphs represent resonance units upon equilibrium (RUEq) of above treatments with mean ± s.e.m. shown from >three runs. GST–hTIM-3 immobilized by amine coupling. Dilutions of hCEACAM1–hTIM-3 single chain, hCEACAM1–hTIM-3 single chain with either blocking hTIM-3-specific peptide, control scrambled peptide, and 26H7 antibody or control MOPC antibody were injected over immobilized GST–hTIM-3 at 25 °C. Flow rate was 25 μl min−1. f, g, 2Fo − Fc maps contoured at 0.9σ showing electron densities for X-ray crystal structure of single chain hCEACAM1–hTIM-3 (PDB code 4QYC). h, Summary of crucial amino acid residues defined biochemically and structurally. ik, Similarity between apo-hCEACAM1 and hTIM-3-associated CEACAM1. Structure of CEACAM1 homodimer at 2.0 Å resolution (PDB code 4QXW) (i). Homophilic ‘YQQN’ concavity indicated consisting of residues Tyr 34, Gln 44, Gln 89 and Asn 97 at hCEACAM1 (IgV)–hCEACAM1 (IgV) interface (j). Superimposition of IgV domain of hCEACAM1 monomer (orange) from i on hCEACAM1 (green) from hCEACAM1–hTIM-3 heterodimer in Fig. 2e (k). Representative of three (be) independent experiments. ***P < 0.001.