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. 2009 Nov 16;187(4):553-67.
doi: 10.1083/jcb.200904149.

The CEACAM1 N-terminal Ig domain mediates cis- and trans-binding and is essential for allosteric rearrangements of CEACAM1 microclusters

Esther Klaile  1 , Olga Vorontsova, Kristmundur Sigmundsson, Mario M Müller, Bernhard B Singer, Lars-Göran Ofverstedt, Stina Svensson, Ulf Skoglund, Björn Obrink
Affiliations

The CEACAM1 N-terminal Ig domain mediates cis- and trans-binding and is essential for allosteric rearrangements of CEACAM1 microclusters

Esther Klaile et al. J Cell Biol. .

Abstract

Cell adhesion molecules (CAMs) sense the extracellular microenvironment and transmit signals to the intracellular compartment. In this investigation, we addressed the mechanism of signal generation by ectodomains of single-pass transmembrane homophilic CAMs. We analyzed the structure and homophilic interactions of carcinoembryonic antigen (CEA)-related CAM 1 (CEACAM1), which regulates cell proliferation, apoptosis, motility, morphogenesis, and microbial responses. Soluble and membrane-attached CEACAM1 ectodomains were investigated by surface plasmon resonance-based biosensor analysis, molecular electron tomography, and chemical cross-linking. The CEACAM1 ectodomain, which is composed of four glycosylated immunoglobulin-like (Ig) domains, is highly flexible and participates in both antiparallel (trans) and parallel (cis) homophilic binding. Membrane-attached CEACAM1 ectodomains form microclusters in which all four Ig domains participate. Trans-binding between the N-terminal Ig domains increases formation of CEACAM1 cis-dimers and changes CEACAM1 interactions within the microclusters. These data suggest that CEACAM1 transmembrane signaling is initiated by adhesion-regulated changes of cis-interactions that are transmitted to the inner phase of the plasma membrane.

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Figures

Figure 1.
Figure 1.
SPR-based analysis of homophilic CEACAM1 ectodomain interactions. Sensorgrams were recorded in a BIAcore 2000 instrument. Rat and human CEACAM1 D(1–4)-Fc proteins were immobilized at a level of 7,100 response units (RU). (A and B) Five concentrations of rat D(1–4)-His (0.36–1.79 g/l, corresponding to 4.4–22.2 µM monomer) were analyzed in HBS/P20/3 mM EDTA (A) or HBS/P20/2 mM Ca2+/2 mM Mg2+ (B). The corrected responses (cRU) for binding to rat D(1–4)-Fc, obtained by subtracting the response in the reference lane (human D(1–4)-Fc), are shown as black data points. The results of global curve fitting to each of the three reaction models (models 1–3; see Results, Materials and methods, and Fig. S1) are shown as residual plots above the sensorgrams. The curve fittings shown in the sensorgrams are according to model 1 (A, purple curves) and model 3 (B, red curves).
Figure 2.
Figure 2.
Particle identification in protein solution and plain buffer. 3D reconstructions of electron micrographs of a 35.4-µM D(1–4) rat CEACAM1 ectodomain solution and plain buffer were achieved by filtered backprojection and COMET refinement. (A) Histogram displaying the volume distribution (given in voxels) of all structures in a reconstructed volume of D(1–4)-containing specimen identified by SWS (1 voxel = 5.74 × 5.74 × 5.74 Å3). The threshold was 1,000 voxels for D(1–4) dimers and 500 voxels for monomers. All volumes <500 voxels were identified as background noise. All but no additional structures that were identified by SWS were also identified by gray-level thresholding. (B) Comparison of two monomer and two dimer particles from the tomogram analyzed in A identified by SWS (top; surface rendered; volumes given in voxels) and gray-level thresholding (bottom; volume rendered). Bars, 5 nm. (C and D) Comparison of the CEACAM1 D(1–4)-His solution (C) and plain buffer (D), both scaled to the same intensity levels by gray-level thresholding. 30-pixel-deep slices (300 × 160 × 30; pixel size, 5.74 Å) of the total reconstructed volumes are shown. Bars, 50 nm.
Figure 3.
Figure 3.
Different presentations of CEACAM crystal structures. (A–C) Published CEACAM PDB data files are presented as ribbon models (top), space-filling models with atomic resolution (middle), and nuclear scattering models at a 20-Å resolution (bottom). The right image of each structure is rotated 90° around the x axis with respect to the corresponding left image. (A) Ectodomain of a mouse CEACAM1 isoform with the D1 domain (top right) fused to the D4 domain (bottom left; PDB accession no. 1L6Z). (B) Two D1 domains of human CEACAM5 (PDB accession no. 2QSQ) in the asymmetric unit (asym unit) in contact via their C″C′CFG faces. (C) Two D1 domains of human CEACAM1 (PDB accession no. 2GK2) in the asymmetric unit in contact via their ABED faces. Bars, 2.5 nm. (D) Ribbon model of the human CEACAM1 D1 domain shown at three different angles to visualize the C″C′CFG and the ABED β sheets.
Figure 4.
Figure 4.
Molecular tomography of CEACAM1 D(1–4)-His and D(2–4)-His ectodomains. Segmentation was performed by gray-level thresholding. (A–K) Molecules are shown unaltered, color coded, and displayed together with schematic models (individual Ig domains are numbered when unambiguously identified). (A–D) Monomeric D(1–4)-His adopting an extended (A and B), kinked (C), or back-folded (D) form. (E) D(1–4)-His dimer interacting exclusively via the D1 domains (C-dimer). (F and G) D(1–4)-His dimers interacting via three (F) or four (G) of their Ig domains (A-dimers). (H) D(1–4)-His trimer consisting of a monomer (green) binding via its D1 domain to an A-dimer (red/blue). Molecules displayed in A–H are presented in 3D in Video 1. (I and J) D(2–4)-His monomers adopting an extended (I) or kinked (J) form. (K) D(2–4)-His parallel dimer. (L) All ectodomains in several reconstructed volumes containing D(1–4)-His in the presence of Ca/Mg (12 vol; 1,091 molecules) or EDTA (14 vol; 888 molecules) and D(2–4)-His in the presence of Ca/Mg (5 vol; 211 molecules) were analyzed. The ectodomains were classified as extended monomers (mono e), kinked plus back-folded monomers (mono k+bf), C-dimers (di C), A-dimers (di A), and trimers (tri). The differences in each ectodomain class compared with D(1–4) (Ca/Mg) populations were analyzed by two-proportion z test. P-values are shown above the histogram bars. Bars, 5 nm.
Figure 5.
Figure 5.
Liposome aggregation mediated by CEACAM1. (A) Aggregation of D(1–4) and D(2–4) proteoliposomes with protein/lipid ratios varying from 1:10 to 1:90 (wt/wt) measured as turbidity at 595 nm at various times after protein addition. (B) 2D electron microscopic images of naked liposomes, D(1–4) proteoliposomes, and D(2–4) proteoliposomes (protein/lipid ratio 1:5). The small black dots indicate 10-nm colloidal gold particles. Bars, 100 nm.
Figure 6.
Figure 6.
Molecular tomography of CEACAM1 ectodomains attached to liposomes. Segmentation was performed by gray-level thresholding. (A) Tomogram of D(1–4)-His–decorated liposomes. The z direction is parallel to the electron beam. (B and C) Free surfaces of D(1–4)-His–decorated (B) and D(2–4)-His–decorated (C) liposomes. (D and E) Monomeric bridges (C-dimers) connecting two liposomes (L1 and L2) are shown at two angles. In the colored panels, D1–D1 contact zones are indicated by a black dotted line, and individual Ig domains are numbered 1–4. Arrowheads point to bridge positions, and arrows point to the binding areas. (F and G) Multimeric bridges connecting two liposomes (L1 and L2) are shown. The bridge in F is composed of seven molecules anchored to L1 (orange) and L2 (green). (G) Two bridging clusters of 6 and ∼10 molecules. (H and I) D(1–4) (n = 130) and D(2–4) (n = 163) monomers/clusters on free-liposome surfaces and D(1–4) monomers/clusters (n = 63) bridging two membranes were analyzed and classified according to size. The differences in each cluster class compared with D(1–4) (free) populations were analyzed by a two-proportion z test. P-values are shown above the histogram bars. Arrows indicate bridge positions. The overviews in B and C and the bridges in E and F are presented in 3D in Videos 2 and 3, respectively. (D–F) Liposome surfaces are indicated by white dashed lines.
Figure 7.
Figure 7.
Cross-linking of CEACAM1 D(1–4)-His and D(2–4)-His. (A and C) Purified D(1–4)-His (A) or D(2–4)-His (C) were cross-linked with BS3 in the presence or absence of 2.5 mM Ca2+, 2.5 mM Mg2+, 3 mM EDTA, and Ni-NTA liposomes at a high protein/lipid ratio (hp; 1:10 [wt/wt]) or a low protein/lipid ratio (lp; 1:90 [wt/wt]) in various combinations. The samples were analyzed by Western blotting for CEACAM1 monomers/dimers/multimers. The black line indicates that intervening lanes have been spliced out. (B) Quantification of D(1–4)-His dimers and higher order multimers. (D) Quantification of D(2–4)-His dimers. Data show mean ± standard deviation of three independent experiments. Statistical analyses (t test) were made with reference to cross-linked protein with no extra additions (Untr.). Significant p-values (α level 0.05) are shown above the histogram bars.
Figure 8.
Figure 8.
A model for adhesion-induced signal generation by CEACAM1. (A and B) CEACAM1 molecules on free-membrane surfaces are organized as a mixture of monomers (A, left), parallel A-dimers (not depicted), and multimers (B, right). Upon adhesion-mediating, trans-homophilic antiparallel C-dimerization, the N-terminal D1 domains undergo conformational changes, which induce formation of parallel cis–A-dimers by an allosteric mechanism. The formation of ectodomain A-dimers is transduced by the transmembrane domains to the cytoplasmic domains, bringing them together in a specific configuration, thereby altering their binding interactions with intracellular signal molecules.
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