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

In this study, we have approached the first step of CEACAM1 transmembrane signaling by analysis of the dynamics and kinetics of the structure and homophilic interactions of the CEACAM1 ectodomain using a combination of surface plasmon resonance (SPR)-based binding analyses, molecular electron tomography, and chemical cross-linking. We found that the CEACAM1 ectodomain is highly flexible, participating in a limited set of structurally well-defined homophilic binding interactions that give rise to two different kinds of dimers as well as trimers and higher order oligomers. When the CEACAM1 ectodomain was associated with liposomal membranes, it became organized in multimeric microclusters with a narrow size distribution. Upon CEACAM1-mediated trans-homophilic membrane adhesion, the level of parallel CEACAM1 cis-dimers increased, and the average number of molecules per cluster decreased. Together, our data provide for the first time evidence for an allostery-based mechanism Figure 1. SPR-based analysis of homophilic CEACAM1 ectodomain interactions. Sensorgrams were recorded in a BIAcore 2000 instrument. Rat and human CEACAM1 D(1-4)-Fc proteins were immobilized at a level of 7,100 response units (RU). (A and B) Five concentrations of rat D(1-4)-His (0.36-1.79 g/l, corresponding to 4.4-22.2 µM monomer) were analyzed in HBS/P20/3 mM EDTA (A) or HBS/P20/2 mM Ca 2+ /2 mM Mg 2+ (B). The corrected responses (cRU) for binding to rat D(1-4)-Fc, obtained by subtracting the response in the reference lane (human D(1-4)-Fc), are shown as black data points. The results of global curve fitting to each of the three reaction models (models 1-3; see Results, Materials and methods, and Fig. S1 ) are shown as residual plots above the sensorgrams. The curve fittings shown in the sensorgrams are according to model 1 (A, purple curves) and model 3 (B, red curves). 555 CEACAM1 N domain regulates cis-and trans-binding • Klaile et al.