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

Cross-linking of liposome-associated CEACAM1 ectodomains at high D(1-4)/liposome ratio (protein/lipid [wt/wt] 1:10) revealed a significant increase in the level of dimers and a decrease of higher order oligomers that was not statistically significant (P = 0.123; Fig. 7, A and B) . At low D(1-4)/liposome ratio (protein/lipid [wt/wt] 1:90), the level of the D(1-4) dimers was similar to that of nonliposome-bound CEACAM1, whereas the higher oligomers disappeared almost completely (Fig. 7, A and B) . The amount of cross-linkable D(2-4) dimers was not changed by liposome anchoring either in the presence or absence of Ca/Mg (Fig. 7, C and D) . These the proportion of A-dimers in the tomograms would mean that some of the parallel dimers that we classified as A-dimers were in fact C-dimers in which the physical binding only was mediated by domain D1. Underestimation of A-dimer formation in the SPR-binding experiments could be caused by homophilic interactions between the immobilized CEACAM1 ectodomains that we could not account for in our SPR-binding algorithms. Another contributing factor might be that the formation of A-dimers, which we treated as one single reaction, is in reality composed of four cooperative reactions in which all of the four Ig domains participate.