Conformational changes of the recombinant extracellular domain of E-cadherin upon calcium binding.
The cell-adhesion protein E-cadherin/uvomorulin exhibits a calcium-dependent homoassociation. The effect of Ca2+ on the extracellular fragment of E-cadherin was studied using the recombinant protein expressed in the baculovirus expression system. The recombinant and native fragment of E-cadherin were found to be similar by many biochemical criteria [Herrenknecht, K. & Kemler, R. (1993) J. Cell Sci. 17, 147-154]. A large and reversible conformational transition was observed upon Ca2+ depletion. A change from a rod-like structure, 22 nm in length, to a more globular assembly of the five subdomains became evident by electron-microscopical analysis. In the presence of Ca2+, the circular dichroic spectra indicated predominantly beta-structure but a more negative ellipticity was observed in the absence of Ca2+. The intrinsic tryptophan fluorescence decreased by 12% upon Ca2+ depletion. Both effects were used for calcium titrations which indicated calcium binding to several sites with average K(d) values of 45-150 microM. Cleavage of the protein fragment by trypsin occurred only at low Ca2+ concentrations and from the calcium-dependence of cleavage rates, a K(d) value of 24 microM was derived. The major site of cleavage was identified by partial sequencing to be located between the two putative calcium-binding sites in the third subdomain from the N-terminus. In agreement with earlier results with the native fragment, the recombinant protein did not associate in the presence or absence of Ca2+. We suggest the calcium-dependent homoassociation therefore depends on additional effects connected with the cell surface association of E-cadherin.
Pokutta
S
S
Biocenter, Department of Biophysical Chemistry, Basel, Switzerland.
Herrenknecht
K
K
Kemler
R
R
Engel
J
J
D000595:Amino Acid Sequence; D000818:Animals; D015820:Cadherins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016624:Negative Staining; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D014357:Trypsin / Q000378:metabolism
D015820:Cadherins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014357:Trypsin; D002118:Calcium