Structural Basis of Adhesion Activation of the Cadherin-Catenin Complex

Thesis (Ph.D.)--University of Washington, 2021E-cadherin is a protein that regulates cell adhesion through adherens junctions. Its dysfunction has implications in development, inflammation, and cancer metastasis. Type I classical cadherins such as E-cadherin are composed of an extracellular domain of 5 extracellular cadherin repeats interspersed with calcium binding domains, followed by a single-pass alpha-helical transmembrane domain, then a C-terminal intrinsically disordered cytoplasmic tail. The tail is bound to alpha-catenin, beta-catenin, and p120-catenin, together known as the cadherin-catenin complex. Through alpha-catenin, the complex is connected to the cytoskeleton. Cadherins form adhesive bonds to neighboring cells through their extracellular domains. These trans-dimers are thought to be formed through a Trp2 strand-swap between EC1 domains of opposing cadherins. A dimer known as the X-dimer is thought to be a short-lived intermediate that leads to the strand-swap dimer. Cadherin dimers are individually weak, but their force is thought to be multiplied when cadherins assemble laterally into adherens junctions. This is also thought to allow for more subtle regulation of adhesion. Previous studies discovered functional monoclonal antibodies (mAbs) that can modulate the activity of E-cadherins, either blocking or activating adhesion between cells. These antibodies not only have the potential for therapeutic benefit, but also can help us to understand the mechanism of regulation of cadherin activation. Cell experiments using activating antibodies to E-cadherin showed that there is a correlation between cadherin activation and p120-catenin dephosphorylation, indicating that there is inside-out regulation of adhesion. However, the mechanism of this is not yet understood. In these studies, we worked toward achieving a better understanding of the molecular basis of the activation of cadherin adhesion. First, we reconstituted the complete and full-length cadherin-catenin complex, including p120-catenin, for the first time. We found that p120-catenin did not interact with the other catenins, and that the whole complex is flexible as a whole. This work can serve as a methodical basis for many other studies looking to examine both the extracellular and intracellular sides of the complex. Second, we focused on the extracellular domain of E-cadherin and examined its dimerization behavior in solution, as well as bound to functional antibody fragments (Fabs). Using cryo-electron microscopy (cryo-EM) we saw that cadherins are far more dynamic than previously predicted, with monomers, X-dimers, strand-swap dimers, and a novel EC4 mediated dimer all strongly represented in 2D class averages. We also found through X-Ray crystallography and cryo-EM that activating Fab 19A11 binding can induce a previously unseen twisted, S-shaped, strand-swap dimer, formed by the N-terminal strand including Trp2 protruding farther outward from each cadherin monomer. This work tells us that cadherin regulation is more dynamic than can easily be observed through crystallography. Finally, in a collaborative project, we sought to gain a biophysical understanding of cadherin activation by 19A11 activating Fab. We saw through atomic force microscopy (AFM) that 19A11 Fab binding enhances the strength of E-cadherin dimers under force - the first such single-molecule experiment demonstrating direct antibody activation of E-cadherin. This was followed up with molecular dynamics (MD) simulations on the crystal structure of 19A11 Fab bound to hE-cadherin EC1-2. These showed that 19A11 Fab enhances cadherin dimerization strength by stabilizing the N-terminal beta strand in the strand-swap dimer confirmation. Taken together, this work brings a dynamic molecular understanding of E-cadherin activation that goes beyond what has previously been observed by primarily static crystallographic studies of isolated extracellular domains. It additionally offers the potential to adapt this framework to better understand other cadherins