Cadherins are homotypic calcium dependent cell adhesion proteins that are essential for embryogenesis as well as adult tissue morphogenesis. However, the molecular details of cadherin interactions are not well understood. It is generally believed that cadherins first form cis (lateral) dimers on the same cell surface via the extracellular regions which act as the basic functional units for cell adhesion. The cis-dimerization in turn initiates cell-cell adhesion via weak trans (adhesive) bond formation which is further strengthened by lateral clustering of cadherin in a cooperative manner. We used single molecule structural (Fluorescence Resonance Energy Transfer) and single molecule functional (Atomic Force Microscopy) assays to test these cadherin binding models. Single molecules labeled with acceptor and donor dyes were immobilized on a substrate and the distance between them was measured using Fluorescence Resonance Energy Transfer. The binding of individual cadherins was also measured using an Atomic Force Microscope. Using these approaches we demonstrate that cadherin cis dimerization is not mandatory for trans interaction and trans dimers can be formed by cadherin monomers interacting via their N-termial EC1 domains. Cis dimerization via outermost EC1 domains are not detected even when cadherins are placed in close proximity by recombinantly dimerizing them at their C-terminus via an antibody Fc fragment. This lateral clustering however enhances the trans binding probability cooperatively. We also demonstrate that Tryptophan at position 2 on the outermost cadherin domain plays an important role in calcium dependent cadherin trans binding. Cadherins are able to form initial encounter complex without swapping the conserved Tryptophan 2. The non-swapped interaction is about 25% the strength of the full exchanged trans dimerization. Tryptophan 2 also prevents non-specific interactions in the absence of Calcium. We suggest that cadherin dimerization proceeds via an induced fit mechanism in which the initial encounter complex undergoes subsequent conformational changes to form the final strand-swapped dimer. This study provides new insights of cadherin interactions at the molecular level, helps resolve the conflicting data regarding trans and cis binding states in literature and may facilitate a better model of cadherin mediated cell-cell adhesion.
|Advisor:||Nelson, W. James|
|School Location:||United States -- California|
|Source:||DAI-B 70/03, Dissertation Abstracts International|
|Subjects:||Cellular biology, Biophysics|
|Keywords:||Cadherin, Cadherin dimers, Cell adhesion, Tryptophan|
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