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 (Kd = 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 Kd of 460 microM for the three bound Ca2+. Assuming a model for three binding sites, which slightly increased the quality of the fit, we obtained two identical Kds of 330 microM and a third much higher Kd 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 Kd of 30 microM. 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.
Koch
A W
AW
Department of Biophysical Chemistry, Biozentrum, University of Basel, Switzerland.
Pokutta
S
S
Lustig
A
A
Engel
J
J
D000818:Animals; D015820:Cadherins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014461:Ultracentrifugation
D015820:Cadherins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D002118:Calcium