Diverse oligomeric states of CEACAM IgV domains

Abstract

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) comprise a large family of cell surface adhesion molecules that bind to themselves and other family members to carry out numerous cellular functions, including proliferation, signaling, differentiation, tumor suppression, and survival. They also play diverse and significant roles in immunity and infection. The formation of CEACAM oligomers is caused predominantly by interactions between their N-terminal IgV domains. Although X-ray crystal structures of CEACAM IgV domain homodimers have been described, how CEACAMs form heterodimers or remain monomers is poorly understood. To address this key aspect of CEACAM function, we determined the crystal structures of IgV domains that form a homodimeric CEACAM6 complex, monomeric CEACAM8, and a heterodimeric CEACAM6-CEACAM8 complex. To confirm and quantify these interactions in solution, we used analytical ultracentrifugation to measure the dimerization constants of CEACAM homodimers and isothermal titration calorimetry to determine the thermodynamic parameters and binding affinities of CEACAM heterodimers. We found the CEACAM6-CEACAM8 heterodimeric state to be substantially favored energetically relative to the CEACAM6 homodimer. Our data provide a molecular basis for the adoption of the diverse oligomeric states known to exist for CEACAMs and suggest ways in which CEACAM6 and CEACAM8 regulate the biological functions of one another, as well as of additional CEACAMs with which they interact, both in cis and in trans.

Keywords: CEACAM; X-ray crystallography; analytical ultracentrifugation; isothermal titration calorimetry.

Fig. 1.

Crystal structure of CEACAM6. (A) The overall structural fold of the IgV domain of CEACAM6 with secondary elements labeled. (B) Structure of the CEACAM6 dimer. (C) Superposition of the CEACAM6 dimer (red chains) onto the CEACAM5 dimer (blue chains). (D) Differences in the dimerization interface result in the loss of π-π stacking (Left), gain of a salt bridge (Center), and loss of a hydrogen bond (Right) for CEACAM6 relative to CEACAM5.

Fig. S1.

Sequence alignment and interface residues of CEACAMs. Sequence alignment of CEACAMs using ClustalW. Secondary structure of CEACAMs are depicted above the alignments. Residues in red are buried in the dimerization interface (CEACAM6/8 and CEACAM8/6 describing residues of CEACAM6 and CEACAM8 in the CEACAM6–CEACAM8 complex, respectively). The number of amino acid exchanges relative to CEACAM5 are shown at the right. Potential glycosylation sites are shown in green.

Fig. 2.

Oligomeric states of homotypic CEACAM preparations. Sedimentation equilibrium analyses (Upper) and residuals of the fits for each curve (Lower) for (A) CEACAM6, (B) CEACAM1, (C) CEACAM5, and (D) CEACAM8. (E) K_dimerization values and SDs for all homotypic CEACAM interactions.

Fig. S2.

Sedimentation equilibrium analyses (Upper) and residuals of fitted data for each curve (Lower) for CEACAM6 mutants (A) I30A, (B) L45A, (C) Q90A and (D) L96A, and CEACAM8 mutants (E) R45A, (F) Q90A, and (G) L96A.

Fig. 3.

Crystal structure of CEACAM8. (A) Crystal structure of CEACAM8 depicting an asymmetrical dimer due to crystal packing relative to CEACAM5 (superimposed onto the CEACAM8 structure shown in gray). (B) An artificial CEACAM8 homodimer modeled by superposition of CEACAM8 onto the CEACAM5 homodimer shows that residues R45 and M97 of CEACAM8 clash in the dimerization interface.

Fig. 4.

ITC binding curve of (A) nonglycosylated CEACAM8 titrated into nonglycosylated CEACAM6 and (B) glycosylated CEACAM8 titrated in to glycosylated CEACAM6.

Fig. S3.

ITC binding curve of (A) CEACAM1 titrated into CEACAM6, (B) CEACAM1 titrated into CEACAM8, (C) CEACAM3 titration into CEACAM6, and (D) CEACAM5 titrated into CEACAM6. (E) Silver-stained SDS/PAGE gel of glycosylated CEACAM6 and CEACAM8 either treated (+) or not treated (−) with PNGaseF.

Fig. 5.

Crystal structure of the CEACAM6/8 complex. (A) Structure of the CEACAM6–CEACAM8 heterodimer. CEACAM6 and CEACAM8 are cyan and green, respectively. (B) Superposition of the CEACAM6–CEACAM8 complex (cyan and green, respectively), CEACAM6 dimer (magenta), and onto the CEACAM5 dimer (gray). (C) 2F_o–F_c composite omit maps of (Left) residues 42–48 of CEACAM6 (cyan), and 93–99 of CEACAM8 (yellow), and (Right) of residues 93–99 of CEACAM6 (cyan) and 42–48 of CEACAM8 (yellow).

Fig. S4.

ITC binding curve of WT CEACAM8 being titrated into (A) CEACAM6 I30A, (B) CEACAM6 L45A, (C) CEACAM6 Q90A, and (D) CEACAM6 L96A. (E) CEACAM8 R45A, (F) CEACAM8 Q90A, and (G) CEACAM8 L96A were titrated against WT CEACAM6.

Fig. 6.

Effects of CEACAM6 homodimers, CEACAM8 monomers and CEACAM6/8 heterodimers on cis and trans interactions. (A) CEACAM6 and CEACAM8 expressed on the same granulocyte energetically favors formation of cis CEACAM6/8 heterodimers. (B) Expression of CEACAM6 and CEACAM8 on epithelial cells and granulocytes, respectively, energetically favors formation of trans CEACAM6/8 heterodimers.