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

Klaile, Esther; Vorontsova, Olga; Sigmundsson, Kristmundur; Müller, Mario M.; Singer, Bernhard B.; Öfverstedt, Lars-Göran; Svensson, Stina; Skoglund, Ulf; Öbrink, Björn

To demonstrate which molecular details that ideally would be observed at a resolution of 20 Å, we show published crystallographic structures of mouse CEACAM1 (Tan et al., 2002) , human CEACAM1 (Fedarovich et al., 2006) , and human CEACAM5 (Fig. 3 , A-C; Korotkova et al., 2008) . The D1 domains of CEACAM1 and CEACAM5 are very similar both in their primary and 3D structures (Watt et al., 2001) . All structures are shown both at atomic resolution (space-filling models), and at 20-Å resolution (nuclear scattering models). The two tandem domains of mouse CEACAM1 (Fig. 3 A) can easily be distinguished at 20-Å resolution. However, it should be noted that the displayed crystallographic structures represent nonglycosylated protein domains, whereas the molecular tomography of rat CEACAM1 in this study was made on highly glycosylated proteins. The tomographic structures are therefore expected to be somewhat larger and not as well resolved. Ig domains have two faces consisting of closely opposed sheets of  strands, CFG, and ABED faces (Fig. 3 D) . Fig. 3 B shows two unglycosylated CEACAM5 D1 domains in close contact at their CFG faces. It is believed that this represents a physiological interaction because mutagenesis experiments have demonstrated that amino acid residues at the CFG surface are involved in homophilic adhesion (Watt et al., 2001) . Fig. 3 C shows two unglycosylated CEACAM1 D1 domains that are in contact via their ABED faces. However, it has been argued that this does not represent a physiological interaction because the ABED surface is highly hydrophobic and is believed to be covered by an oligosaccharide attached to Asn70 (Tan et al., 2002; Fedarovich et al., 2006) . Both of the structures (Fig. 3 , B and C) are compact to as C-dimers because their structure and shape can easily account for antiparallel intermembrane adhesion, which is mediated by mutual D1-D1 binding between the CFG -sheet faces (Fig. 3 B ; Watt et al., 2001) . Another type of D(1-4) dimers, referred to as A-dimers, was characterized by in-register, close binding between three or all four of the Ig domains (Fig. 4 , F and G). Because the liposome adhesion experiments (see the dominant species (45.2% and 56.1% of the molecules, respectively). In addition to the monomers, several complexes of homophilically bound D(1-4) ectodomains occurred, as judged both from the shapes and dimensions/volumes of the recorded particles. The most common complex type was fully or partly extended dimers held together only by reciprocal binding between the largest end domains (Fig. 4 E) . They were referred and EDTA, respectively. This shows that both types of dimers can form in the presence and absence of divalent cations, but that both dimerization reactions were enhanced by the divalent cations, which agrees with the SPR data. Furthermore, the homophilic binding affinity leading to C-dimer formation was significantly higher than that for A-dimer formation. This suggests that the C-dimers and A-dimers correspond to the type 1 and type 2 dimers recorded in the SPR-binding experiments, respectively, because the equilibrium concentrations of type 1 dimers were significantly higher than those of type 2 dimers.