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

Abstract

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.

Structure of the extracellular region of the adhesion GPCR CELSR1 reveals a compact module which regulates G protein-coupling

“…56 One side of the bead model is thinner than the other, in agreement with multiple reports of a hinge region between CADH5 and CADH6. 56,57…”

“…Using the deposited crystal structure of human CELSR1 CADH4-7 (PDB 7SZ8), we investigated the dynamics of the proposed hinge region within the cadherin repeat region of CELSR. 56,57 In CELSR1, two of the canonical acidic calcium coordinating residues between CADH5 and CAHD6 are found as K533 and T564 (Figure 4 A, B). These substitutions prevent the coordination of two out of three Ca 2+ ions canonically found between cadherin repeats.…”

“…Our data provide molecular detail to a previous report showcasing low resolution atomic force microscopy images of the full CELSR2 extracellular region. 56 The CADH9-GAIN CMM is large, with a solvent-accessible surface area of 74,000 Å 2 and presents several putative ligand binding sites to the extracellular milieu, including the site on the LamG2 domain. It remains to be defined how the known PCP ligands Frizzled and Van Gogh interact with CELSR1 in cis.…”

“…This study also shows that the CADH repeats likely wrap around each other tightly, using multiple cadherin domains to form tight extended dimer species similar in principle to protocadherins. 56 Another recent study determined crystal structures of CELSR1 CADH1-4 and CADH4-7, giving the first high-resolution insight into the CADH repeat region of CELSR and revealing a non-canonical linker between CADH5 and CADH6. 57 Next, CELSRs have a series of epidermal growth factor (EGF) and Laminin G (LamG) domains.…”

In Situ Real-Time Observation of Photoinduced Nanoscale Azo-Polymer Motions Using High-Speed Atomic Force Microscopy Combined with an Inverted Optical Microscope

“…High-speed atomic force microscopy (HS-AFM) is a well-recognized tool for capturing biological dynamic motions, including conformational changes in proteins with high spatiotemporal resolutions, enabled by recent technical developments such as small cantilevers, fast scanners, and dynamic feedback control. It has contributed significantly to numerous biological discoveries by providing movies of molecular dynamics at the single-molecule level, including walking myosin V, rotary motion of rotorless F 1 -ATPs, DNA cleavage by CRISPR-Cas9, etc. − It has already become an indispensable technique in the field of biology.…”