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

A closer analysis of the data showed that the tomography gave a 1.7-fold higher degree of monomers and a 0.7-fold lower degree of total dimers than the SPR-binding experiments in the presence of Ca/Mg. In addition, the differences in the equilibrium constants and the proportion of C-and A-dimers obtained by the two methods suggest that the proportion of A-dimers might have been overestimated in the tomography experiments and/or that the formation of A-dimers might have been underestimated in the SPR-binding determinations. Overestimation of participate in dimer formation in Ig domains (Edmundson et al., 1975) . However, a simultaneous interaction of two areas in the same Ig domain has, to our knowledge, only previously been suggested for the adhesion molecules JAM-1 (Kostrewa et al., 2001) , NCAM (Soroka et al., 2003) , and TAG-1 (Mörtl et al., 2007) . The binding site in the CEACAM1 D1 domain that mediates trans-homophilic binding, and presumably operates in C-dimer formation, has been localized to the CFG face by site-directed mutagenesis (Watt et al., 2001) . The exact location of the other binding site in relation to the CFG face is unknown and could not be determined from the electron tomograms. Although crystallographic structures showing domains in close contact do not necessarily reflect a biologically relevant association, the finding of two unglycosylated CEACAM1 D1 domains in the asymmetric crystal unit that interact hydrophobically across the ABED faces (Fig. 3 C; Fedarovich et al., 2006) might indicate that this could represent the second binding site. However, in the crystal structure of mouse CEACAM1, this surface was covered by the oligosaccharide N linked to Asn-70 (Tan et al., 2002) , and therefore, it has been argued that the ABED surface of CEACAM N-terminal domains cannot participate in physiological homophilic binding (Fedarovich et al., 2006) . However, such a scenario should not be completely ruled out because oligosaccharides are flexible units, and it might be possible that the ABED surface would transiently and dynamically be exposed for participation in protein-protein interactions. Because CEACAM1 is variably glycosylated in different cell types and tissues (Odin et al., 1988; Singer et al., 2002) , glycosylation might even represent a mechanism to control its putative potential to form A-dimers.