STRUCTURAL BASIS OF CALCIUM INDUCED E-CADHERIN RIGIDIFICATION AND DIMERIZATION Bhushan Nagar,^* Michael Overduin,⁺ Mitsuhiko Ikura,⁺ James M. Rini^{,* *}Departments of Molecular and Medical Genetics and Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada, ⁺Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario, M5G 2M9, Canada.

The cadherins mediate cell adhesion and play a fundamental role in normal development. They typically consist of five tandemly repeated extracellular domains, a single membrane spanning segment, and a cytoplasmic region. The N-terminal extracellular domains mediate cell-cell contact while the cytoplasmic region interacts with the cytoskeleton through the catenins. Cadherins are defined by their calcium dependence: removal of calcium abolishes adhesive activity and renders cadherins vulnerable to proteases. The two N-terminal domains of epithelial cadherin (E-cadherin) have been expressed in E. coli and crystallized from 11mg/mL solutions by the hanging-drop vapour-diffusion method in the presence of Ca²⁺. The well solution consisted of 1.2M Ammonium sulphate, 30 mM CaCl₂, 100mM Tris buffer pH 9.0. Plate-like crystal clusters of dimensions 0.6 x 0.4 x .02 mm grow within 2 days in space group C2 (a=122.0, b=80.5, c=73.2 Å, ß=114.5deg.. The structure was solved (to 2.0 Å resolution) using a combination of multiwavelength anomalous diffraction (MAD) phasing and real space averaging techniques. The structure reveals a two-fold symmetric dimer, each molecule of which binds a contiguous array of three bridged calcium ions. Each molecule of the dimer is composed of two 7-stranded ß-barrels connected by a 10 residue linker. Not only do the bound calcium ions linearize and rigidify the molecule, but they promote dimerization. The E-cadherin dual domain structure defines the role played by calcium in the cadherin mediated formation and maintenance of solid tissues.