Calcium Binding and Homoassociation of E-cadherin Domains

• Published in: Biochemistry (Easton) 1997-06, Vol.36 (25), p.7697-7705
• Main Authors: Koch, Alexander W, Pokutta, Sabine, Lustig, Ariel, Engel, Jürgen
• Format: Article
• Language: English
• Subjects: Animals; Cadherins - genetics; Cadherins - isolation & purification; Cadherins - metabolism; Calcium - metabolism; Circular Dichroism; Cloning, Molecular; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; Mice; Molecular Sequence Data; Protein Binding; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Ultracentrifugation
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9705624

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.