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Mechanism of E-cadherin dimerization probed by NMR relaxation dispersion

Significance Dimerization of transmembrane protein epithelial cadherin (E-cadherin) molecules extending from apposing cells is a critical event in calcium-dependent cell adhesion, a process essential for the formation and maintenance of tissues and organs. This study elucidates the molecular mechanism of E-cadherin dimerization by utilizing nuclear magnetic resonance spectroscopy to detect and characterize sparsely populated molecular species. In this mechanism, E-cadherin monomers first interact at an auxiliary interface to form a dimeric intermediate, which is followed by the conformational changes required to form the mature dimer. Besides contributing to the understanding of E-cadherin binding and recognition, the mechanism described in this work may illustrate a more general mechanism by which proteins with occluded binding sites interact with each other. Epithelial cadherin (E-cadherin), a member of the classical cadherin family, mediates calcium-dependent homophilic cell–cell adhesion. Crystal structures of classical cadherins reveal an adhesive dimer interface featuring reciprocal exchange of N-terminal β-strands between two protomers. Previous work has identified a putative intermediate (called the “X-dimer”) in the dimerization pathway of wild-type E-cadherin EC1–EC2 domains, based on crystal structures of mutants not capable of strand swapping and on deceleration of binding kinetics by mutations at the X-dimer interface. In the present work, NMR relaxation dispersion spectroscopy is used to directly observe and characterize intermediate states without the need to disrupt the strand-swapped binding interface by mutagenesis. The results indicate that E-cadherin forms strand-swapped dimers predominantly by a mechanism in which formation of a weak and short-lived X-dimer–like state precedes the conformational changes required for formation of the mature strand-swapped dimeric structure. Disruption of this intermediate state through mutation reduces both association and dissociation rates by factors of ∼104, while minimally perturbing affinity. The X-dimer interface lowers the energy barrier associated with strand swapping and enables E-cadherins to form strand-swapped dimers at a rate consistent with residence times in adherens junctions.