Calcium Binding and Homoassociation of E-cadherin Domains

Cadherins are single pass transmembrane glycoproteins which mediate calcium dependent cell−cell adhesion by homophilic interactions. To reveal the molecular details of calcium binding and homoassociation, we recombinantly expressed in Escherichia coli a domain pair consisting of the first two domains of E-cadherin (ECAD12) and the single domains 1, 2, and 5. ECAD12 encompasses the most N-terminal of the four putative calcium-binding pockets in the extracellular region of E-cadherin. Equilibrium dialysis experiments revealed that the single domains do not bind Ca2+, but ECAD12 was found to bind three calcium ions. ECAD12 dimerizes (K d = 0.08 ± 0.02 mM) in the presence of Ca2+ as we could demonstrate by analytical ultracentrifugation. Calcium binding to ECAD12 induces conformational changes which were monitored by electrophoretic mobility and by circular dichroism. By analyzing our equilibrium dialysis data with a single binding site model, we found an average K d of 460 μM for the three bound Ca2+. Assuming a model for three binding sites, which slightly increased the quality of the fit, we obtained two identical K ds of 330 μM and a third much higher K d of 2 mM. The entire extracellular region of E-cadherin, which was recombinantly expressed in mammalian cells, binds nine Ca2+ with a much lower average K d of 30 μM. Therefore, we conclude that the four calcium binding pockets are not identical. Since binding to ECAD12 occurs at Ca2+ concentrations close to those in the extracellular space, we suggest that the N-terminal domain pair might be involved in calcium regulation of E-cadherin mediated cell−cell adhesion.