An ensemble of flexible conformations underlies mechanotransduction by the cadherin-catenin adhesion complex

Abstract

The cadherin-catenin adhesion complex is the central component of the cell-cell adhesion adherens junctions that transmit mechanical stress from cell to cell. We have determined the nanoscale structure of the adherens junction complex formed by the α-catenin•β-catenin•epithelial cadherin cytoplasmic domain (ABE) using negative stain electron microscopy, small-angle X-ray scattering, and selective deuteration/small-angle neutron scattering. The ABE complex is highly pliable and displays a wide spectrum of flexible structures that are facilitated by protein-domain motions in α- and β-catenin. Moreover, the 107-residue intrinsically disordered N-terminal segment of β-catenin forms a flexible "tongue" that is inserted into α-catenin and participates in the assembly of the ABE complex. The unanticipated ensemble of flexible conformations of the ABE complex suggests a dynamic mechanism for sensitivity and reversibility when transducing mechanical signals, in addition to the catch/slip bond behavior displayed by the ABE complex under mechanical tension. Our results provide mechanistic insight into the structural dynamics for the cadherin-catenin adhesion complex in mechanotransduction.

Keywords: adherens junction; mechanotransduction; negative stain electron microscopy; small-angle X-ray scattering; small-angle neutron scattering.

Fig. 1.

Conformational variability of the ABE complex revealed by negative stain EM. (A) Amino acid sequence and domain boundaries of EcadCT, α-catenin, and β-catenin. Previous studies showed that residues 783 to 882 in EcadCT bind to the armadillo repeat domain of β-catenin at residues 134 to 662 (23) and that residues 57 to 145 in the N domain of α-catenin bind to β-catenin at residues 118 to 148 (24). (B and C) Gel filtration profile (B) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel of the reconstituted ABE complex (C). (D) Electron micrograph area of negatively stained ABE complex. (Scale bar: 50 nm.) (E) Selected class averages of negatively stained ABE complex obtained with the ISAC procedure (SI Appendix, Fig. S1 shows all averages). Particles are shown from a more compact conformation starting in the upper left to a more extended conformation in the lower right. The arrowheads in the first average indicate the apparent hinge points around which the domains can move. Side length of individual averages: 31.2 nm.

Fig. 2.

Overall structure of the ABE complex. (A and B) SEC-SAXS and 3D shape of the ABE complex. Scattering intensity I(q) plot of the ABE complex (A). SI Appendix, Fig. S2 shows the SEC-SAXS profile, Guinier plot, and P(r) of the ABE complex. The 3D shape of the ABE complex reconstructed from the SAXS data using the program Gasbor (68) (B). The red line shown in A is the fit to generate the 3D shape. (C–F) Composite structure of the selectively deuterated ^dA^hB^hE complex from contrast variation SANS analysis. Contrast variation SANS data of ^dA^hB^hE in 0, 20, 42, 60, 90, and 100% (vol/vol) D₂O buffer (C). P(r) functions of the ^dA^hB^hE complex at different contrasts (D); C shows quality of fit. Composite 3D shape of the ABE complex generated from the contrast variation SANS data using the program Monsa (37) (E). SI Appendix, Fig. S3A shows quality of fit to the scattering data when generating the 3D shape. Atomic models of α-catenin (from Monte Carlo simulation shown in Fig. 3G), β-catenin (from Monte Carlo simulation shown in Fig. 4F), and the cadherin cytoplasmic domain (PDB ID code 1I7W) were docked into the envelopes using the program UCSF Chimera (72). Green, deuterated α-catenin; magenta, hydrogenated β-catenin and EcadCT.

Fig. 3.

Conformation of α-catenin by itself and as part of the ^dA^hB^hE complex. (A) SEC-SAXS data of the α-catenin monomer alone in solution (black dots). Data are taken from ref. . SANS data of the ^dA^hB^hE complex, c = 3.7 mg/mL, in 42% D₂O (red squares) at the contrast-matching point of ^hB^hE, which only reveals the conformation of ^dα-catenin in the complex. Red lines are fits to the experimental scattering data for generating P(r). SI Appendix, Fig. S4 A and B shows Guinier plots and Kratky plots. (B) P(r) of the α-catenin monomer in solution (black) and of ^dα-catenin in the ^dA^hB^hE complex (red) generated from the scattering data in A. (C) The 3D shapes of the α-catenin monomer generated from SEC-SAXS data using the program DAMMIF/DAMMIN (73). (D) The 3D shape of ^dα-catenin within the ^dA^hB^hE complex generated from contrast-matching SANS data in 42% D₂O using the program Gasbor (68). Docked into the 3D shapes are the α-catenin structures in solution and in the ^dA^hB^hE complex, which were generated by Monte Carlo simulations using the program SASSIE (43). (E and F) R_g distribution (E) and D_max distribution (F) from EOM analysis (41, 42) of the SAXS and SANS data of the α-catenin monomer in solution (black) and of ^dα-catenin as a part of the ^dA^hB^hE complex (red). For the α-catenin monomer, R_flex = 75.3% (pool 84.5%) and R_σ = 0.68. For ^dα-catenin within the ^dA^hB^hE complex, R_flex = 72.9%, Pool 85.4%, and R_σ = 0.62. These values suggest that α-catenin is a flexible molecule. (G) Flexible structural models of ^dα-catenin in the ^dA^hB^hE complex obtained from Monte Carlo simulations show that the M domain and the ABD can adopt multiple configurations. The simulations were performed using the SANS data of ^dA^hB^hE in 42% D₂O buffer as constraints. SI Appendix, Fig. S4 C and D shows quality of fit.

Fig. 4.

Conformation of β-catenin by itself and as part of the ^hB^dE or ^dA^hB^dE complex. (A) SEC-SAXS data of β-catenin (black squares; reveal the conformation of β-catenin alone in solution) and contrast-matching SANS data of the ^hB^dE complex in 100% D₂O buffer (red squares; reveal the conformation of ^hβ-catenin in ^hB^dE) and of the ^dA^hB^dE complex in 100% D₂O buffer (blue squares; reveal the conformation of ^hβ-catenin in the ^dA^hB^dE complex). The lines are fits to the experimental data for generating the P(r) functions. SEC-SAXS profile of β-catenin in solution is shown in SI Appendix, Fig. S6. (B) P(r) of β-catenin in solution (black) and as part of the ^hB^dE complex (red) or the ^dA^hB^dE complex (blue). (C) Dimensionless Kratky plots of the SAXS and SANS data shown in A. (D–F) The 3D molecular shape of β-catenin in solution from SEC-SAXS (D) and β-catenin in the ^hB^dE complex from SANS in 100% D₂O (E) and in the ^dA^hB^dE complex (F) from SANS in 100% D₂O buffer. The 3D shapes were generated using the program Gasbor (68).

Fig. 5.

The intrinsically disordered N-terminal segment of β-catenin participates in ABE complex assembly. (A) The binding affinity of the BE complex for full-length α-catenin is reduced if β-catenin lacks the N-terminal 107 amino acid residues (B_N-107E). (B) Comparing the binding affinities of the BE complex for the M domain, the ABD of α-catenin, and the full-length α-catenin (K_d values are in the text). (C–E) SPR sensorgrams for the binding of the BE complex to full-length α-catenin (C), to the M domain (D), and to the ABD (E) of α-catenin. The red lines are fit to a 1:1 kinetic binding model to obtain the association and dissociation rate constants k_a and k_d, respectively, shown in Table 2.

Fig. 6.

Structural models of the ABE complex from SAXS and SANS. (A) Fit of the structural models generated by Monte Carlo simulations using SASSIE (43) to the SEC-SAXS data of the ABE complex and (B) to the SANS data of the ^dA^hB^hE complex in 0% D₂O buffer; χ² vs. R_g of the fits of the computed models to the experimental data are shown in A and B. (C) Representative structural models of the ABE complex that are consistent with the SAXS and SANS data. To comply with the binding data shown in Fig. 5, the N-terminal 107 amino acid residues have to be in contact with the M domain or ABD of α-catenin.