Spontaneous Calcium-Independent Dimerization of the Isolated First Domain of Neural Cadherin.

Cadherins are calcium-dependent, transmembrane adhesion molecules that assemble through direct noncovalent association of their N-terminal extracellular modular domains. As the transmembrane component of adherens junctions, they indirectly link adherent cells' actin cytoskeletons. Here, we investigate the most distal extracellular domain of neural cadherin (N-cadherin), a protein required at excitatory synapses, the site of long-term potentiation. This domain is the site of the adhesive interface, and it forms a dimer spontaneously without binding calcium, a surprising finding given that calcium binding is required for proper physiological function. A critical tryptophan at position 2, W2, provides a spectroscopic probe for the "closed" monomer and strand-swapped dimer. Spectroscopic studies show that W2 remains docked in the two forms but has a different apparent interaction with the hydrophobic pocket. Size-exclusion chromatography was used to measure the levels of the monomer and dimer over time to study the kinetics and equilibria of the unexpected spontaneous dimer formation ( Kd = 130 μM; τ = 2 days at 4 °C). Our results support the idea that NCAD1 is missing critical contacts that facilitate the rapid exchange of the βA-strand. Furthermore, the monomer and dimer have equivalent and exceptionally high intrinsic stability for a 99-residue Ig-like domain with no internal disulfides ( Tm = 77 °C; Δ H = 85 kcal/mol). Ultimately, a complete analysis of synapse dynamics requires characterization of the kinetics and equilibria of N-cadherin. The studies reported here take a reductionist approach to understanding the essential biophysics of an atypical Ig-like domain that is the site of the adhesive interface of N-cadherin.