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

The homophilic binding properties of CEACAM1 ectodomains were analyzed by SPR-based flow cell biosensor analysis. D(1-4) and D(2-4) CEACAM1 ectodomain Fc fusion proteins were immobilized as ligands on a BIAcore chip, and both His-tagged ( Fig. 1) and Fc fusion ectodomains (not depicted) were used as soluble analytes. The rat D(1-4) proteins bound specifically to immobilized rat D(1-4) ( Fig. 1 ) but not to rat D(2-4) (not depicted). No explicit binding of the rat D(2-4) constructs was observed either to rat D(1-4) or rat D(2-4) ligands (unpublished data). Thus, the recordable homophilic binding must be caused by D1-D1 interactions. The D(1-4) binding was characterized by rapid on and off rates in the presence of both EDTA and Ca/Mg, but the extent of binding was larger in Ca/Mg (Fig. 1 ). In addition, the divalent cations induced a more complex binding pattern, with a slower binding superimposed on the dominant rapid association/ dissociation, demonstrating that at least two different binding reactions occurred. The Ca/Mg effect was not influenced by the His tag because the same divalent cation dependence was seen when CEACAM1 Fc fusion proteins, which lack a His tag, were used as analytes (unpublished data).