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

Binding experiments were performed with a BIAcore 2000 instrument (GE Healthcare). Rat and human CEACAM1 D(1-4)-Fc and rat CEACAM1 D(2-4)-Fc were immobilized on a CM5 sensor chip by amine coupling at pH 4.0. All interaction analyses were performed at 25°C at a flow rate of 20 µl/min in 0.15 M NaCl, 10 mM Hepes (Hepes-buffered saline [HBS]), pH 7.4, and 0.005% P20 detergent supplemented with either 2 mM CaCl 2 /2 mM MgCl 2 or 3 mM EDTA. Histagged rat CEACAM1 ectodomains were injected for 3 min followed by buffer injection for 5 min. After each binding cycle, the chip surface was regenerated with two cycles of 4 M LiCl/HBS and one cycle of 0.25% P20/HBS. Corrected binding profiles (sensorgrams) for rat CEACAM1 ectodomains were obtained by subtracting the response in the reference lane (human CEACAM1-Fc) from the response in the binding lane (rat CEACAM1-Fc). Data modification including scale transformation and background subtraction was performed with the BIAevaluation program (version 3.1). Algorithms for global curve fitting to the different reaction models were written in IGOR Pro (version 6.0; WaveMetrics, Inc.). The basic equations and parameters used for mass transport calculations were described previously . Detailed descriptions of the reaction models and the equations underlying the curve fit algorithms are provided in Fig. S1 and the following paragraph. A molecular mass of 81 kD and a frictional ratio (f/f 0 ) of 1.256 were used for D(1-4)-His. Varying the frictional ratio between 1.256 and 1.43 did not result in any significant differences of the association/dissociation constants.