Sequence and structural determinants of strand swapping and binding affinity in cadherin domains

Abstract

Cadherins are cell surface adhesion proteins important for tissue development and maintenance of tissue integrity. Type I and type II, or "classical", cadherins form adhesive dimers via an interface that involves the exchange, or "swapping", of the N-terminal β-strands from their membrane-distal EC1 domains. These interactions are highly specific; cadherins that are very similar in sequence display different binding properties. Here we ask which sequence and structural features in EC1 domains are responsible for β-strand swapping and for differences in binding affinity. We first create a comprehensive database consisting of multiple alignments of each type of cadherin domain. We use the known three-dimensional structures of classical cadherins to identify conserved positions in multiple sequence alignments that appear to be crucial determinants of the cadherin domain structure. We further identify features that are unique to EC1 domains and implicated in swapping, and we verify the contributions of these structural elements through experiments that modify the swapping potential of different cadherin domains.

To study binding affinity, we analyze the E-cadherin and N-cadherin interfaces computationally to generate a set of candidate positions that may be the source of differences in cadherin binding affinities. Analytic ultracentrifugation and surface plasmon resonance experimental studies of mutant cadherins demonstrate the influence of specific sites on homophilic and heterophilic binding, providing a molecular dissection of the E-cadherin and N-cadherin interfaces. These results indicate that subtle changes in the swapping interface region are responsible for the observed differences in binding behavior, and that changes in electrostatic interactions at the periphery of the interface can modulate binding affinities.

Lastly, we present a computational method for identifying proteins that swap domains and apply the method to the Protein Data Bank (PDB) to build a large database of swapped proteins. We characterize the swapped interfaces present in the database and compare them to a reference set of non-swapping interfaces.

Our studies of cadherins indicate that it is possible to identify specific structural determinants that may facilitate swapping. The swapping database lays the groundwork for applying this approach to other families of swapping proteins, which will further elucidate the evolution of this phenomenon.