Structural basis of αE-catenin-F-actin catch bond behavior

Abstract

Cell-cell and cell-matrix junctions transmit mechanical forces during tissue morphogenesis and homeostasis. α-Catenin links cell-cell adhesion complexes to the actin cytoskeleton, and mechanical load strengthens its binding to F-actin in a direction-sensitive manner. Specifically, optical trap experiments revealed that force promotes a transition between weak and strong actin-bound states. Here, we describe the cryo-electron microscopy structure of the F-actin-bound αE-catenin actin-binding domain, which in solution forms a five-helix bundle. In the actin-bound structure, the first helix of the bundle dissociates and the remaining four helices and connecting loops rearrange to form the interface with actin. Deletion of the first helix produces strong actin binding in the absence of force, suggesting that the actin-bound structure corresponds to the strong state. Our analysis explains how mechanical force applied to αE-catenin or its homolog vinculin favors the strongly bound state, and the dependence of catch bond strength on the direction of applied force.

Keywords: actin; adherens junction; alphae-catenin; catch bond; cryo-EM; molecular biophysics; none; structural biology; vinculin.

Figure 1.. α-Catenin in adherens junctions.

(A) Schematic of AJ and the role of αE-catenin in the connection to the actin cytoskeleton. The extracellular region of cadherins (green) bind to one another between cells, and their cytoplasmic domains bind to β-catenin (yellow). β-Catenin binds to α-catenin (pink/red), which binds to F-actin (orange) weakly in the absence of force (top panel). Tension (indicated by arrows) favors the strong actin-binding state of α-catenin, and also produces conformational changes in α-catenin that lead to recruitment of vinculin (light blue). While the net direction of the force is likely perpendicular to the junction (black arrows), there will be local force components along the mixed-polarity filaments toward their pointed (-) ends through actomyosin contractility (grey arrows). (B) Primary structure of αE-catenin; binding sites for β-catenin, vinculin and F-actin are indicated. (C) Crystal structure of αE-catenin ABD (Ishiyama et al., 2018) (PDB 6dv1); the five helices H1-H5, the N-terminal capping helix H0, and the C-terminal extension (CTE) are labeled.

Figure 2.. Cryo-EM analysis of the αE-catenin ABD–F-actin complex.

(A) Cryo-EM map of the actin-ABD structure. The segmented ABDs are shown in magenta. The (-) end of the filament is shown at the top, and the (+) end at the bottom. (B) Molecular model of a section of an actin filament bound to αE-catenin ABDs, same orientation as (A) and with transparent density map overlaid. Actin protomers are colored according to their long-pitch helix in blue and yellow. The bound ABDs are shown in magenta and pink. (C, D) Closeups of model and cryo-EM map showing residues on H4 (panel C) and the CTE (panel D) that have been studied by site-directed mutagenesis. The ABD is shown in red, and two monomers of actin in different shades of blue. In (D), a neighboring copy of the ABD along the filament is shown in pink. Actin residue labels are italicized.

Figure 2—figure supplement 1.. Cryo EM analysis.

(A, B) Representative micrographs of actin filaments in the presence of truncated 671–906 ABD (A) and full-length ABD (B). Segments recognized as bare are marked with red dots, segments recognized as having ABD bound are marked with cyan dots. Scale bars are 50 nm. (C) Local resolution analysis. (D) Fourier Shell Correlation (FSC) curves using two reconstructions independently derived from two halves of the data. The 0.143 FSC cutoff used for estimating the resolution is indicated as a dashed line.

Figure 2—figure supplement 2.. Representative gels and binding curves for actin co-sedimentation assays with αE-catenin N-terminal deletion constructs.

Pellets of actin co-sedimentation assays with (+A) and without (-A) F-actin are shown. * Indicates the actin band and the arrow indicates the ABD band.

Figure 2—figure supplement 3.. Close-up of αE-catenin–F-actin interactions.

Colors are as shown in Figure 2. Hydrogen bonds are shown as dashed lines, and van der Waals contacts as solid lines. (A) Interactions of the αE-catenin CTE. (B) Interaction of αE-catenin K842 actin residues H87 and Y91. (C) αE-catenin K797 salt bridge with actin E334.

Figure 2—figure supplement 4.. Representative gels and binding curves for actin co-sedimentation assays with αE-catenin C-terminal deletion constructs.

Pellets of actin co-sedimentation assays with (+A) and without (-A) F-actin are shown. * Indicates the actin band and the arrow indicates the ABD band.

Figure 2—figure supplement 5.. Alignment of α-catenin and vinculin ABD sequences.

(A) Residues that contact actin are shown in stick representation and colored in dark and light blue according to which actin protomer they contact, as shown Figure 2. The hydrophobic cluster residues W705, Y837 and W859 are shown in light orange. (B) ABD sequence alignments. The secondary structure elements of the unbound ABD and the actin-bound ABD are shown above the alignments. Residues that contact actin and the hydrophobic cluster residues are highlighted in the colors used in panel A. Residues in grey form the interface between H1 and H2/H5. The sequence alignment was done in Geneious 10.2.2 (www.geneious.com). The figure was prepared with ENDscript (Robert and Gouet, 2014) and UCSF Chimera (Pettersen et al., 2004). Abbreviations used M.mus.-Mus musculus D.rer. - Danio rerio, D.mel. – Drosopila melanogaster, C. ele. - Caenorhabditis elegans N.vec. - Nematostella vectensis.

Figure 3.. Overall changes in ABD structure upon binding to F-actin.

Comparison of the unbound ABD crystal structure (PDB 6dv1; light orange) with the ABD in the actin-bound state (magenta). (A) Overall comparison; the orientation is rotated approximately 180° from that shown in Figure 1c. (B) Top view of H0 packing interactions lost upon its removal, and rearrangements of helices 2–5. The left panel depicts packing interactions of H0 residues I672, M673 and L676 with H5 residues V809, G811 and A815 (all highlighted in orange), and the right panel overlay shows the resulting changes in H4 and H5.

Figure 3—figure supplement 1.. Differences in the CTE free and bound to F-actin.

In the actin-bound state, the aromatic cluster of W705 in H1, Y837 in H5, and W859 in the CTE repacks due to the shift in position of the remaining turn of H1, which pulls W705 away from Y837 and W859 and is replaced by M861 of the CTE. Colors are the same as in Figure 3.

Figure 3—figure supplement 2.. Comparison of free and actin-bound αE-catenin and vinculin ABDs.

The orientations are the same as in Figure 3A. (A) The free vinculin (slate blue; PDB 1qkr Bakolitsa et al., 1999) and αE-catenin (light orange; PDB 6dv1 Ishiyama et al., 2018) ABDs superimpose with an RMSD of 1.2 Å. (B) The actin-bound vinculin (grey; PDB 3jb1 Kim et al., 2016) and αE-catenin (magenta) ABDs superimpose with an RMSD of 1.3 Å. (C) Superposition of the free (slate blue) and actin-bound vinculin (grey) ABDs, RMSD = 1.5 Å.

Figure 4.. Stability of the H1- H2/H5 interface.

(A) Time course of elastase digestion of αE-catenin 671–906. The two smaller fragments analyzed by N-terminal sequencing are indicated with asterisks. (B) Rainbow diagram of the unbound αE-catenin ABD (PDB 6dv1), colored as in Figure 1c. Residue labels for the H5 (red) helix are shown in white for clarity. The two residues at the elastase cut sites are indicated in gold. Side chains in the H1- H2/H5 interface are shown in stick representation.

Figure 5.. Model of the weak and strong actin-binding states of αE-catenin.

(A) Superposition of the isolated αE-catenin ABD on the actin-bound structure reveals no major clashes with F-actin (left panel). When the ABD is bound to F-actin, H0 and H1 dissociate from the H2-5 bundle, which results in the extension and shift of the C-terminal part of H4 as well as ordering and repositioning of the CTE to bind to actin. (B) Schematic diagram of αE-catenin ABD conformational states when unbound, weakly bound, and strongly bound to actin. Cooperative binding of the ABD, as observed in the cryo-EM structure, is illustrated for the strong state. Note that in the strong state, the H0 and H1 regions are drawn as helices when dissociated from the H2-5 bundle, but it is likely that they are unstructured in this case (see text for details).

Figure 5—figure supplement 1.. Clash of isolated ABD structure with actin.

The isolated ABD structure (light orange; PDB 6dv1) was superimposed on the actin-bound structure (magenta). Actin is shown in blue. αE-catenin residues K683 (H1), E799 (H4-H5 connector), and D813 and M816 (H5) clash with actin residues K328, I330 and P333. Comparison with the actin-bound ABD shows that small movements associated with at least partial removal of H1 would relieve these clashes.

Figure 6.. Model of directional catch bonding.

Actin and the αE-catenin ABD in the strong and weak states are illustrated as in Figure 5B. The N-terminus of the ABD is shown tethered to a stationary point, that is, as part of the cadherin/β-catenin/α-catenin complex. The grey arrows indicate the direction of force. (A) Tension applied to the bound strong state prevents re-binding of H0/H1 to the H2-H5 bundle. Force applied in the (-) direction will move the H1 sequence away from the H2-H5 bundle and place this region in an unfavorable orientation for rebinding, whereas force directed in the (+) direction will place the H1 sequence closer to and in a more favorable orientation for rebinding. See Video 1 for an animated version. (B) Tension applied to the bound weak state will remove H0/H1 from the H2-H5 bundle. Force applied in the (-) direction will tend to pull H0/H1 away from the H2-H5 bundle, whereas force in the (+) direction is predicted to have a smaller effect on H0/H1 dissociation. See Video 2 for an animated version.