Multiple dimeric structures and strand-swap dimerization of E-cadherin in solution visualized by high-speed atomic force microscopy

Classical cadherins play key roles in cell-cell adhesion. The adhesion process is thought to comprise mainly two steps: X-dimer and strand-swap (SS-) dimer formation of the extracellular domains (ectodomains) of cadherins. The dimerization mechanism of this two-step process has been investigated for type I cadherins, including E-cadherin, of classical cadherins, whereas other binding states also have been proposed, raising the possibility of additional binding processes required for the cadherin dimerization. However, technical limitations in observing single-molecule structures and their dynamics have precluded the investigation of the dynamic binding process of cadherin. Here, we used high-speed atomic force microscopy (HS-AFM) to observe full-length ectodomains of E-cadherin in solution and identified multiple dimeric structures that had not been reported previously. HS-AFM revealed that almost half of the cadherin dimers showed S- (or reverse S-) shaped conformations, which had more dynamic properties than the SS- and X-like dimers. The combined HS-AFM, mutational, and molecular modeling analyses showed that the S-shaped dimer was formed by membrane-distal ectodomains, while the binding interface was different from that of SS- and X-dimers. Furthermore, the formation of the SS-dimer from the S-shaped and X-like dimers was directly visualized, suggesting the processes of SS-dimer formation from S-shaped and X-dimers during cadherin dimerization.

Automated summary

Classical cadherins are important in cell-cell adhesion. High-speed atomic force microscopy (HS-AFM) was used to observe full-length ectodomains of E-cadherin and discovered previously unreported dimeric structures, with about half of the cadherin dimers showing S-shaped conformations and greater dynamics than SS and X-like dimers. Mutational and molecular modeling analyses revealed that the S-shaped dimer had a different binding interface from SS and X-dimers. The formation of SS-dimer from S-shaped and X-like dimers was directly visualized.