Binding partner- and force-promoted changes in \u03b1E-catenin conformation probed by native cysteine labeling

Sabine Pokutta,William I Weis,Yee S Kee,Ksenia Terekhova,Jing Li,Emad Tajkhorshid,Alexander R Dunn,Gerald Fuller

doi:10.1038/s41598-019-51816-3

Abstract

Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via β-catenin and the F-actin binding protein αE-catenin. When subjected to mechanical force, the cadherin•catenin complex can tightly link to F-actin through αE-catenin, and also recruits the F-actin-binding protein vinculin. In this study, labeling of native cysteines combined with mass spectrometry revealed conformational changes in αE-catenin upon binding to the E-cadherin•β-catenin complex, vinculin and F-actin. A method to apply physiologically meaningful forces in solution revealed force-induced conformational changes in αE-catenin when bound to F-actin. Comparisons of wild-type αE-catenin and a mutant with enhanced vinculin affinity using cysteine labeling and isothermal titration calorimetry provide evidence for allosteric coupling of the N-terminal β-catenin-binding and the middle (M) vinculin-binding domain of αE-catenin. Cysteine labeling also revealed possible crosstalk between the actin-binding domain and the rest of the protein. The data provide insight into how binding partners and mechanical stress can regulate the conformation of full-length αE-catenin, and identify the M domain as a key transmitter of conformational changes.

Highlights

Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via β-catenin and the F-actin binding protein αE-catenin
The structure of αE-catenin comprises a series of α-helical bundles organized into three main regions: an N-terminal (N) domain that consists of two four-helix bundles (NI and NII) that both binds β-catenin and mediates homodimerization; a middle (M) domain consisting of three four-helix bundles (MI, MII, and MIII) that harbor binding sites for vinculin and other protein partners; and a C-terminal, five-helix bundle termed the
We developed a novel method to directly probe the effect of mechanical force on αE-catenin by examining changes in cysteine accessibility under physiologically relevant levels of mechanical load induced by fluid shear

Summary

Introduction

Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via β-catenin and the F-actin binding protein αE-catenin. In this study, labeling of native cysteines combined with mass spectrometry revealed conformational changes in αE-catenin upon binding to the E-cadherinβ-catenin complex, vinculin and F-actin. The middle (M) domain comprises three 4 helix bundles, MI (dark green), MII (light green) and MIII (orange); vinculin binds to the two central helices in MI (shown in pink) The F-actin binding domain (ABD) is shown in brown; the position of the ABD differs in the two copies in PDB 4IGG26, and chain B is shown here. We examine the conformational dynamics and inter-domain coupling of αE-catenin associated with its binding to cadherinβ-catenin, F-actin, and vinculin by measuring changes in native cysteine accessibility in solution. Combined with structural and thermodynamic data, these experiments provide direct experimental data for structural changes associated with allosteric regulation of αE-catenin

Methods

Results

Conclusion