Thermodynamic stability of domain 2 of epithelial cadherin

Cadherin is a cell adhesion molecule that participates in ordered calcium-dependent self-association interactions both between molecules on the same cell surface (cis-interactions) and on neighboring cell surfaces (trans-interactions). Cadherin is a transmembrane protein that has 3-7 independently folded beta-barrel extracellular domains. Both types of self-association interactions are mediated through the most N-terminal domain (Domain 1). Although the structural nature of the trans-interactions is clear, the nature of the cis-interactions is ambiguous despite several high-resolution structural studies. From earlier studies, it is understood that for the trans-interactions to happen, cis-interactions are mandatory. Hence, our first steps are to study the energetic driving forces for the cis-interactions. We have simplified the approach by first examining participating extracellular domains individually. We report here our initial experiments into the stability of Domain 2 of E-cadherin (ECAD2). ECAD2 appears monomeric, according to results from mass spectrometry and sedimentation equilibrium studies. We report denaturation data from differential scanning calorimetric experiments, and temperature and denaturant-induced unfolding experiments monitored by circular dichroism. These studies give a unified picture of the energetics of ECAD2-folding and stability, for which DeltaG degrees is 6.6 kcal/mol, T(m) is 54 degrees C, DeltaH(m) is 90 kcal/mol, and DeltaC(p) is 1300 cal/Kmol. These parameters are independent of calcium up to 5 mM, indicating that ECAD2 does not bind calcium at physiological calcium levels.