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Fig. 5. Binding of mAb GC4 to dimeric N-cadherin-Fc mutants in the presence or absence of calcium. (a) The wild type (Wt) and the W2G mutant are compared in the presence of 1.25 mM Ca²⁺. (b) The same titration was performed in the absence of Ca²⁺; the two titrations were performed together in the same assay plate and values can be compared directly. Results in the presence of EGTA (not shown) were almost identical to those in b. (c) Wild-type N-cadherin-Fc was tested at varying levels of calcium. (d) The hydrophobic pocket mutant A78M was compared with wild-type N-cadherin in the presence of 1 mM calcium. This comparison was also made in the absence of calcium (e). Similarly, a comparison between the N-terminal extension mutant MDP and wild type N-cadherin was made in the presence (f) or absence (g) of 1 mM calcium. Finally, coordination of calcium in the domain 1-2 junction was disrupted using the mutation D134A, while retaining calcium (1 mM) in the assay buffer. (h) The effect of this mutation, compared with the wild type. (i) The greater effect of D134A on the MDP extension mutant.

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