title: Sequential binding of calcium leads to dimerization in neural cadherin.
abstract: Neural cadherin (N-cadherin) is a calcium-dependent homophilic cell-adhesive molecule and critical for synaptogenesis and synapse maintenance. The extracellular region plays an important role in cadherin-mediated cell adhesion and has five tandemly repeated ectodomains (EC1-EC5) with three calcium-binding sites situated between each of these domains. Adhesive dimer formation is significantly dependent on binding of calcium such that mutations in the calcium-binding sites adversely affect cell adhesion. To investigate the relative significance of the calcium-binding sites at the EC1-EC2 interface in calcium-induced dimerization, we mutated three important amino acids, D134, D136, and D103, in NCAD12, a construct containing EC1 and EC2. Spectroscopic and chromatographic experiments showed that all three mutations affected calcium binding and dimerization. Mutation of D134, a bidentate chelator in site 3, severely impaired the binding of calcium to all three sites. These findings confirm that binding to site 3 is required for binding to occur at site 2 and site 1. Interestingly, while the D103A mutation diminished only the affinity for calcium, it completely eliminated dimerization. Equilibrium dialysis experiments showed a stoichiometry of 3 at 2 mM calcium for D103A, but no dimerization was apparent even at 10 mM calcium. These results indicate that calcium binding alone is not sufficient for dimerization but requires cooperativity between calcium-binding sites. In summary, our findings confirm that the calcium-binding sites are occupied sequentially in the order of site 3, then site 2 and site 1, and that cooperativity between site 2 and site 1 is essential for formation of adhesive dimers by N-cadherin.
authors.affiliation: Department of Chemistry and Biochemistry, University of Mississippi, University, Mississippi 38677, United States.
authors.forename: Nagamani
authors.initials: N
authors.lastname: Vunnam
authors.forename: Susan
authors.initials: S
authors.lastname: Pedigo
mesh_terms: D000465:Algorithms; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D015820:Cadherins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013816:Thermodynamics
chemical_list: D015820:Cadherins; D011994:Recombinant Proteins; D002118:Calcium