Date of Award
Ph.D. in Chemistry
Chemistry and Biochemistry
Cadherins are calcium-dependent molecules that play essential roles in embryonic development, tissue morphogenesis, and cancer progression. Epithelial (E-) cadherin is present at adherens junctions, structures that are important for maintaining tissue integrity. At adherens junctions, cell–cell adhesion complexes are formed as clusters of cadherin dimers through strand-swapped dimerization between two interacting protomers from adherent cells. Recently, a number of studies of dimer formation of E-cadherin supported a two-step adhesive binding model. First, an inter-molecular initial encounter complex (X-dimer) forms at the linker region of the first two extracellular domains, EC1 and EC2. Next, strand-swap dimerization occurs via exchange or ''swap'' of the βA-strands at the EC1 interface between two E-cadherin protomers from opposing cells. In this new model, the formation of the transient X-dimer intermediate juxtaposes the EC1 domains of interacting protomers to facilitate the formation of a mature strand-swapped adhesive dimer.
Our interest is in understanding the molecular components in E-cadherin that are responsible for fast dimer assembly and disassembly. Particular interest is residue K14, which is proposed to have an interaction with D138 on the partner protomer, an ionic interaction that stabilizes the X-dimer interface. While the wild type protein displays behavior characteristic of a rapidly exchanging monomer-dimer equilibrium, the mutant K14E exhibits slow exchange kinetics presumably due to electrostatic repulsion between E in position 14 and D138. Current studies investigate whether it is the loss of K in position 14, or the gain of E that creates the kinetic barrier to dimer: monomer exchange.
In this study, we investigated the link between Ca2+-dependent disassembly kinetics of an X-interface mutant by studying three mutants at position 14, K14A, K14E, and K14S. We measured the effect of these mutations on the structure and function of E-cadherin. Thermal denaturation studies showed that these mutants have identical stability as found for WT protein. The results of size exclusion chromatography indicated that the constructs had similar dimerization dissociation constants to WT (∼100 μM; K14S- Kd =155 ± 12 μM; K14A- Kd = 143 ± 10 μM; K14E- Kd = 91 ± 13 μM). Similarly, the calcium binding affinity was identical to WT for these mutants. These data imply that K14 is not required for equilibrium properties E-cadherin. However, as evident from size exclusion chromatography, all three K14 mutants exhibited slow dimer exchange, a result that conclusively indicates that K14 is required for rapid dimerization kinetics observed in WT E-cadherin.
Howard, Xiaoyun Z., "Contribution of specific amino acids to calcium-dependent dimerization of epithelial cadherin" (2017). Electronic Theses and Dissertations. 1363.
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