Calcium-dependent homoassociation of E-cadherin by NMR spectroscopy: changes in mobility, conformation and mapping of contact regions

J Mol Biol. 2002 Dec 6;324(4):823-39. doi: 10.1016/s0022-2836(02)01137-3.

Abstract

Cadherins are calcium-dependent cell surface proteins that mediate homophilic cellular adhesion. The calcium-induced oligomerization of the N-terminal two domains of epithelial cadherin (ECAD12) was followed by NMR spectroscopy in solution over a large range of protein (10 microM-5 mM) and calcium (0-5 mM) concentrations. Several spectrally distinct states could be distinguished that correspond to a calcium-free monomeric form, a calcium-bound monomeric form, and to calcium-bound higher oligomeric forms. Chemical shift changes between these different states define calcium-binding residues as well as oligomerization contacts. Information about the relative orientation and mobility of the ECAD12 domains in the various states was obtained from weak alignment and 15N relaxation experiments. The data indicate that the calcium-free ECAD12 monomer adopts a flexible, kinked conformation that occludes the dimer interface observed in the ECAD12 crystal structure. In contrast, the calcium-bound monomer is already in a straight, non-flexible conformation where this interface is accessible. This mechanism provides a rational for the calcium-induced adhesiveness. Oligomerization induces chemical shift changes in an area of domain CAD1 that is centered at residue Trp-2. These shift changes extend to almost the entire surface of domain CAD1 at high (5 mM) protein concentrations. Smaller additional clusters of shift perturbations are observed around residue A80 in CAD1 and K160 in CAD2. According to weak alignment and relaxation data, the symmetry of a predominantly dimeric solution aggregate at 0.6 mM ECAD12 differs from the approximate C2-symmetry of the crystalline dimer.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Cadherins / chemistry*
  • Cadherins / isolation & purification
  • Cadherins / metabolism*
  • Calcium / chemistry
  • Calcium / metabolism*
  • Carbon Isotopes
  • Crystallography, X-Ray
  • Diffusion
  • Dimerization
  • Escherichia coli / metabolism
  • Hydrogen / chemistry
  • Mice
  • Models, Molecular*
  • Molecular Sequence Data
  • Nitrogen Isotopes
  • Nuclear Magnetic Resonance, Biomolecular
  • Proline / chemistry
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Rotation
  • Solutions
  • Tryptophan / chemistry

Substances

  • Cadherins
  • Carbon Isotopes
  • Nitrogen Isotopes
  • Solutions
  • Hydrogen
  • Tryptophan
  • Proline
  • Calcium