Structural and Thermodynamic Characterization of Cadherin·β-Catenin·α-Catenin Complex Formation

Sabine Pokutta,Hee-Jung Choi,Goran Ahlsen,Scott D Hansen,William I Weis

doi:10.1074/jbc.m114.554709

Abstract

The classical cadherin·β-catenin·α-catenin complex mediates homophilic cell-cell adhesion and mechanically couples the actin cytoskeletons of adjacent cells. Although α-catenin binds to β-catenin and to F-actin, β-catenin significantly weakens the affinity of α-catenin for F-actin. Moreover, α-catenin self-associates into homodimers that block β-catenin binding. We investigated quantitatively and structurally αE- and αN-catenin dimer formation, their interaction with β-catenin and the cadherin·β-catenin complex, and the effect of the α-catenin actin-binding domain on β-catenin association. The two α-catenin variants differ in their self-association properties: at physiological temperatures, αE-catenin homodimerizes 10× more weakly than does αN-catenin but is kinetically trapped in its oligomeric state. Both αE- and αN-catenin bind to β-catenin with a Kd of 20 nM, and this affinity is increased by an order of magnitude when cadherin is bound to β-catenin. We describe the crystal structure of a complex representing the full β-catenin·αN-catenin interface. A three-dimensional model of the cadherin·β-catenin·α-catenin complex based on these new structural data suggests mechanisms for the enhanced stability of the ternary complex. The C-terminal actin-binding domain of α-catenin has no influence on the interactions with β-catenin, arguing against models in which β-catenin weakens actin binding by stabilizing inhibitory intramolecular interactions between the actin-binding domain and the rest of α-catenin.

Highlights

Cadherin cell adhesion molecules are linked to the actin cytoskeleton by the proteins ␤- and ␣-catenin
We investigated quantitatively and structurally ␣E- and ␣N-catenin dimer formation, their interaction with ␤-catenin and the cadherin1⁄7␤-catenin complex, and the effect of the ␣-catenin actin-binding domain on ␤-catenin association
␣-Catenin Dimerization and the ␣-Catenin/␤-Catenin Interaction—We characterized the self-association properties of ␣E- and ␣N-catenins by sedimentation equilibrium analytical ultracentrifugation (AUC). ␣E-catenin homodimerizes with a Kd of ϳ25 ␮M at 25 and 37 °C (Fig. 2 and Table 2)

Summary

Background

Cadherin cell adhesion molecules are linked to the actin cytoskeleton by the proteins ␤- and ␣-catenin. We investigated quantitatively and structurally ␣E- and ␣N-catenin dimer formation, their interaction with ␤-catenin and the cadherin1⁄7␤-catenin complex, and the effect of the ␣-catenin actin-binding domain on ␤-catenin association. The biochemical and structural properties of ␣-catenin indicate that it is a dynamic, conformationally complex protein that serves as a hub for assembly of cell-cell junctions and in the regulation of actin dynamics. To understand the molecular basis of its regulated interactions, we have quantitatively and structurally analyzed homodimerization, ␤-catenin binding, and ternary cadherin1⁄7␤-catenin1⁄7␣-catenin complex formation of mammalian ␣E- and ␣N-catenins. We show that both ␣-catenin variants bind identically to ␤-catenin and to the. We show that the ␣-catenin ABD has no influence on binding to ␤-catenin, which argues against an allosteric model in which the ABD has inhibitory intramolecular interactions with the rest of ␣-catenin that are enhanced by ␤-catenin binding

EXPERIMENTAL PROCEDURES

RESULTS

Findings

DISCUSSION