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. b– e, 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 (RU Eq) 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, 2 F o − F c maps contoured at 0.9 σ 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. i– k, 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 ( b– e) independent experiments. *** P < 0.001.