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

Because the input concentration of the CEACAM1 D(1-4) ectodomain was known, the molar concentrations of monomers, C-dimers, A-dimers, and trimers could be calculated from their relative abundance (Fig. 4 L) . Accordingly, it was possible to determine the equilibrium dissociation constants for C-and A-dimer formation by applying the law of mass action. The K D for the monomer/C-dimer binding was 43.8 µM and 72.1 µM in Ca/Mg and EDTA, respectively. The K D for the monomer/A-dimer binding was 113 µM and 260 µM in Ca/Mg CEACAM1 D(1-4) bridges were identified in the contact areas between adjacent, adhering liposomes (Fig. 6 , D-G). 34 different contact areas having a total of 63 bridges were analyzed. The number of bridges per contact area varied from one to five. The bridges occurred both as single antiparallel dimers formed from monomers attached to the respective membranes (Fig. 6 , D and E; and Video 3) and as clusters containing several D(1-4) ectodomains (Fig. 6 , F and G; and Video 3). Highly organized zipper-like structures were not observed. In some monomeric bridges, the individual Ig domains were resolved, and the ectodomains were kinked to variable degrees, demonstrating that the inherent inter-Ig domain flexibility persisted (Fig. 6, D and E; and Video 3). In many bridges, a large part of the individual ectodomains was oriented parallel to the lipid membrane ( Fig. 6 D) . However, in the cluster bridges, it was not possible to unambiguously distinguish individual molecules as a result of the dense packing.