Crystal structures of Drosophila N-cadherin ectodomain regions reveal a widely used class of Ca 2+ -free interdomain linkers

Vertebrate classical cadherins mediate selective calcium-dependent cell adhesion by mechanisms now understood at the atomic level. However, structures and adhesion mechanisms of cadherins from invertebrates, which are highly divergent yet function in similar roles, remain unknown. Here we present crystal structures of three- and four-tandem extracellular cadherin (EC) domain segments from Drosophila N-cadherin (DN-cadherin), each including the predicted N-terminal EC1 domain (denoted EC1’) of the mature protein. While the linker regions for the EC1’-EC2’ and EC3’-EC4’ pairs display binding of three Ca 2+ ions similar to that of vertebrate cadherins, domains EC2’ and EC3’ are joined in a “kinked” orientation by a previously uncharacterized Ca 2+ -free linker. Biophysical analysis demonstrates that a construct containing the predicted N-terminal nine EC domains of DN-cadherin forms homodimers with affinity similar to vertebrate classical cadherins, whereas deleting the ninth EC domain ablates dimerization. These results suggest that, unlike their vertebrate counterparts, invertebrate cadherins may utilize multiple EC domains to form intercellular adhesive bonds. Sequence analysis reveals that similar Ca 2+ -free linkers are widely distributed in the ectodomains of both vertebrate and invertebrate cadherins. Taken together, our findings provide a plausible answer to an intriguing question in cell adhesion: How do large cadherins containing numerous extracellular cadherin domains fit into intercellular spaces of comparable dimensions to those where the five-domain vertebrate classical cadherins mediate cell adhesion? Our results suggest that the unique Ca 2+ -free linkers observed in the structures of DN-cadherin ectodomain fragments and also found to be present in a large number of nonclassical cadherins may impart complex “folded” ectodomain architectures to these cadherins to facilitate their specific biological functions. These findings provide an important step forward in understanding cadherin-mediated cell adhesion in invertebrates, and also yield new insights into the structure and function of the numerous vertebrate cadherins with large ectodomains. Based on the unique Ca 2+ -free interdomain linkage seen in the DN-cadherin crystal structures, we performed bioinformatic analyses of the entire cadherin superfamily. Within the set of 23,340 cadherin domains identified in 3,673 proteins, we found 2,504 linkers with different combinations of missing Ca 2+