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 gain further insights into the mechanism of CEACAM1mediated adhesion, we investigated His-tagged rat CEACAM1 ectodomains anchored to nickel-nitrilotriacetic acid (Ni-NTA) liposomes. D(1-4), but not D(2-4), induced adhesion between liposome vesicles, as demonstrated both by turbidity measurements ( Fig. 5 A) and 2D electron microscopy ( Fig. 5 B) . The inability of D(2-4) to mediate adhesion confirms previously published data (Wikström et al., 1996) that domain D1 mediates trans-homophilic binding and that antiparallel binding involving domains D2, D3, and D4 does not occur. The rate and extent of D(1-4)-mediated liposome aggregation increased with increasing protein/liposome ratio (Fig. 5 A) . Electron tomography revealed the presence of CEACAM1 ectodomains both on freeliposome surfaces and in contact areas between adjacent vesicles (Fig. 6 ). D(1-4) ectodomains on free-liposome surfaces occurred as a mixture of monomers, dimers, and clusters containing 3-12