Neural Cadherin: A Study of Domain 1.

Cadherins are a class of transmembrane proteins responsible for calcium dependent cell-cell adhesion. Classic cadherins share a common structure involving five extracellular domains, each connected by a 7 residue sequence known as a linker region. As these domains are modular in nature, this study focuses primarily on the first extracellular domain of neural cadherin without and with the adjacent linker region. NCADl and NCADl-Ll. Upon calcium binding, a tryptophan residue located in domain 1 allows for the adhesive activity of the cadherin through formation of a strand crossover structure. With the dissected NCADl and NCADl-LI, the binding pocket for calcium is not complete, so dimerization is not expected for these constructs. However, we did observe the two constructs in a dimeric form that was not in exchange with monomer. This dimer was converted to monomer by heating the proteins to 37 °C. Structural and thermal characterization experiments were performed on both constructs in the apo state and in the presence of calcium and salt to compare this domain with other neural cadherin dissections. The addition of calcium had no spectral consequences on either of the two domains, while NCADl-LI was observed to become spectrally similar to NCADl with the addition of salt. We found that both constructs unfold at an uncharacteristically high temperature with a midpoint of ~ 74 °C. This irregularity may be attributed to the formation of a plaque-type structure which gives the false sense of high thermal stability. Formation of this unusual plaque structure was reversible, and high salt concentrations abrogated plaque formation for NCADl-LI. Results for domain 1 constructs were very similar to results from studies for the two domain construct, NCAD12, and contrasted sharply with that obtained from domain 2 studies presented elsewhere. In summary, these studies are the first thermodynamic studies of domain 1 from a member of the classical cadherin family. Interpretation of results from thermal denaturation studies was complicated by formation of this unusual plaque structure. Interestingly, this plaque is formed reversibly and is a characteristic of neural cadherin constructs that contain domain 1