Regulation of multiple dimeric states of E-cadherin by adhesion activating antibodies revealed through Cryo-EM and X-ray crystallography

Abstract

E-cadherin adhesion is regulated at the cell surface, a process that can be replicated by activating antibodies. We use cryo-EM and X-ray crystallography to examine functional states of the cadherin adhesive dimer. This dimer is mediated by N-terminal beta strand-swapping involving Trp2, and forms via a different transient X-dimer intermediate. X-dimers are observed in cryo-EM along with monomers and strand-swap dimers, indicating that X-dimers form stable interactions. A novel EC4-mediated dimer was also observed. Activating Fab binding caused no gross structural changes in E-cadherin monomers but can facilitate strand swapping. Moreover, activating Fab binding is incompatible with the formation of the X-dimer. Both cryo-EM and X-ray crystallography reveal a distinctive twisted strand-swap dimer conformation caused by an outward shift in the N-terminal beta strand that may represent a strengthened state. Thus, regulation of adhesion involves changes in cadherin dimer configurations.

Molecular mechanism for strengthening E-cadherin adhesion using a monoclonal antibody

“…Since residue D58 on the 19A11 heavy chain forms a salt bridge with K14 on Ecad, it is likely that 19A11 binding blocks X-dimer formation. In agreement with this suggestion, a recent cryogenic electron microscopy study shows that 19A11 selectively interacts with Ecad strand-swap dimers and does not bind to X-dimers (20). While blocking X-dimer formation could potentially prevent strand-swap dimer dissociation and thus strengthen adhesion, our data suggest that the force-induced dissociation of strand-swap dimers do not involve an X-dimer intermediate.…”

“…Previous structural studies ( 19 ) show that similar to wild-type (WT) Ecad, the K14E mutant interacts via strand–swap dimerization. Recent size–exclusion chromatography studies have shown that similar concentrations of 19A11 bind to both K14E mutant and WT Ecad ( 20 ). However, 19A11 does not recognize denatured Ecad in Western blots ( SI Appendix , Fig.…”

“…2E, dashed green and dashed orange lines). The average RMSF for Ecads in the presence and absence of the antibody showed that 19A11 binding reduces the rmsf of its corresponding binding regions on Ecad (residues [13][14][15][16][17][18][19][20]. The binding of 19A11 also stabilizes the two adjoining regions, the β-strand (residues 1-12) and the E13-R99 and K14-D58.…”

“…However, our study does not address whether X-dimers play a role in the force-free dissociation of the 19A11–Ecad complex or examine the role of X-dimers in the dissociation of the 19A11–Ecad complex on the cell surface. Furthermore, a recent study shows that Ecad bound to 19A11 adopts a twisted conformation which may potentially impact the stability of strand–swap dimers ( 20 ). Finally, in addition to binding in trans conformations, neighboring Ecads on the same cell surface also form cis dimers ( 26 ).…”

Observation of E-cadherin Adherens Junction Dynamics with Metal-Induced Energy Transfer Imaging and Spectroscopy

“…Initially, this X-dimer was identified through sit e-directed mutations in E-cad (18, 19). More recently, the X-dimer was finally observed in wild-type cadherins using cryo-electron microscopy (20).…”