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

Although homophilically binding proteins have been analyzed by flow cell biosensor techniques (Korotkova et al., 2008) , it has previously not been possible to analyze the kinetics of such a system because no algorithms have existed that can deal with a situation in which the same binding interactions take place both within the mobile phase and between the mobile and solid phases. However, the novel curve-fitting algorithms that we present in this study allowed us to identify two different reactions that resulted in the formation of two types of dimers and one type of trimer in the presence of divalent cations. The equilibrium dissociation constants that were obtained from the curve fitting were in good agreement with the equilibrium dissociation constants determined from the tomography data. Both methods gave constants that were of the same order of magnitude for either type of dimerization reaction and showed the same dependence on divalent cations. These findings strongly support the interpretations of both the SPR-binding analyses and the molecular electron tomography.