An autoinhibited structure of α-catenin and its implications for vinculin recruitment to adherens junctions

Abstract

α-Catenin is an actin- and vinculin-binding protein that regulates cell-cell adhesion by interacting with cadherin adhesion receptors through β-catenin, but the mechanisms by which it anchors the cadherin-catenin complex to the actin cytoskeleton at adherens junctions remain unclear. Here we determined crystal structures of αE-catenin in the autoinhibited state and the actin-binding domain of αN-catenin. Together with the small-angle x-ray scattering analysis of full-length αN-catenin, we deduced an elongated multidomain assembly of monomeric α-catenin that structurally and functionally couples the vinculin- and actin-binding mechanisms. Cellular and biochemical studies of αE- and αN-catenins show that αE-catenin recruits vinculin to adherens junctions more effectively than αN-catenin, partly because of its higher affinity for actin filaments. We propose a molecular switch mechanism involving multistate conformational changes of α-catenin. This would be driven by actomyosin-generated tension to dynamically regulate the vinculin-assisted linkage between adherens junctions and the actin cytoskeleton.

Keywords: Adherens Junction; Cadherins; Catenin; Cell Adhesion; Cytoskeleton; Vinculin; α-Catenin.

FIGURE 1.

Crystal structure of αEcatNM in the autoinhibited conformation. A, α-catenin architecture. α-Catenin contains three major domains: N, M, and C. Structured regions are indicated by colored boxes with αE-catenin residue numbers shown below. Vinculin contains five domains, D1–D5. α-Catenin shares ∼30% sequence identity with vinculin at Vinculin Homology regions, VH1, VH2, and VH3. B, 6.5-Å structure of αEcatNM. The VBS consists of the second and third α-helices (indicated by single and double asterisks, respectively) of the M_I bundle. C, homodimer structure of the αEcatNM. One protomer is outlined by a cyan dashed line. Two chains of αE-catenin form an asymmetric dimer with two juxtaposed M_I bundles. D, the unfurled structure of the M_I region bound to the D1 domain of vinculin (PDB code 4EHP; M_I in black and D1 in gray) (30) is aligned to the fourth helix of the M_I bundle (green) in the autoinhibited state. E, close-up view of the four-helix bundle formed by the M_I region. Amino acid residues in the hydrophobic core of the M_I bundle are shown as sticks. F, numerous salt bridges formed between charged residues (sticks) from different regions are involved in the stabilization of the M_I-M_III and M_II-M_III interfaces. The location of these interfaces is indicated by a magenta box in B. G, GST-vinculin-D1/GST pulldown assay with wild type αE-catenin or its single residue mutants, M371W, E521A, and R551A. The M371W mutant was designed to directly disrupt the M_I bundle (Fig. 1E).

FIGURE 2.

Crystal structures of the αE-catenin M_II-III fragment. A, crystal structure of the M_II-III fragment within αEcatNM. Angles between the M_II and M_III bundles are shown in A–C. B, crystal structure of the M_II-III fragment within the M fragment residues 377–633 (PDB code 1H6G) (25). C, crystal structure of the M_II-III fragment within the M fragment residues 385–651 (PDB code 1L7C) (28). The location of Arg⁵⁵¹, which is in close proximity with Asp⁵⁰³ to form salt bridges at the M_II-M_III interface in the αEcatNM structure (Fig. 1F), is shown as cyan sticks. D, previously determined structures of M_II-III fragments (PDB codes 1H6G and 1L7C) are superposed onto the M_II-III structure of αEcatNM by aligning the M_II bundles. The M_III bundles in PDB codes 1H6G and 1L7C are further rotated 124° and 93°, respectively, away from where the M_III bundle is located in αEcatNM, possibly due to the absence of N domain and M_I bundle. E, superposition of the M domain structure of αEcatNM and the D3–D4 domain structure of vinculin (PDB code 1ST6).

FIGURE 3.

Junctional organization in R2/7 cells stably transfected with various αE-catenin (A) or αN-catenin (B) mutants tagged with EGFP, or transfected with EGFP only as a control. Schematic figures represent a series of mutant constructs used. Cells were triple-immunostained for GFP, vinculin, and ZO-1 (a tight junction protein). αEcatFL, full-length αE-catenin; αEcatNM_I, αE-catenin-NM_I fragment; αEcatNM_I-III, αE-catenin-NM_I-III fragment; and αEcatNM_I-III/αNcatC, a chimera consisting of αE-catenin-NM_I-III fragment fused to αN-catenin-C fragment. αNcatFL, full-length αN-catenin; αNcatNM_I, αN-catenin-NM_I fragment; αNcatNM_I-III, αN-catenin-NM_I-III fragment; and αNcat NM_I-III/αEcatC, a chimera consisting of αN-catenin-NM_I-III fragment fused to the αE-catenin-C fragment. Scale bars, 10 μm.

FIGURE 4.

Crystal structure of the C domain of αN-catenin. A, actin pelleting assays with αE-catenin C domain (αEcatC) and αN-catenin C domain (αNcatC). Supernatant (S) and pellet (P) fractions are indicated. B, the average percentage (and S.D.) of total protein cosedimenting with F-actin in three independent experiments was: αEcatC, 20.0(±2.6)%; αNcatC, 12.0(±2.0)%. C, crystal structure of αNcatC consists of six α-helices and a short β-hairpin and forms a five-helix bundle. Disordered N and C termini extensions are indicated by dashed lines. Regions equivalent to upper and lower actin-binding sites of vinculin (59)(supplemental Fig. S7B) are indicated. D and E, close-up views of the “upper site” (D) and “lower site” (E) of αN-catenin. Side chains of non-identical residues between αE-catenin (blue) and αN-catenin (green for conserved residues and red for non-conserved residues) are shown as sticks. F, structural alignment of actin-binding domain sequences of αN-catenin, αE-catenin, and vinculin (PDB code 1ST6). αE- and αN-catenins share 87% identical and 93% similar residues within their C domain sequences. αE-catenin and vinculin residues that are identical to αN-catenin residues are indicated by dots. Non-identical residues of αE-catenin are shown in blue, whereas αN-catenin residues in green and red indicate conserved and non-conserved substitutions found between αE- and αN-catenins. Vinculin residues that constitute the upper and lower sites (59) are shown as orange and magenta, respectively. Secondary structure information (green bar for α-helix and red arrow for β-strand) based on the crystal structures of αNcatC is shown.

FIGURE 5.

Full-length structure of αN-catenin. A, sedimentation equilibrium analysis of αN-catenin. αN-catenin exists as a monodisperse species with an average molecular mass of 97.4 kDa, which is approximately the size expected of a monomer. In contrast, αE-catenin exists in a monomer-dimer equilibrium (K_d = 73 μm) with an average molecular mass of 132.3 kDa. B, actin pelleting assay with full-length αN-catenin (αNcat). C, experimental scattering curve (red) of αN-catenin and a fitted theoretical scattering curve (blue) (χ² = 1.4) of its full-length structure model (supplemental Fig. S9A). D, docked crystal structures of αEcatNM and αNcatC within the ab initio molecular envelope of full-length αN-catenin (gray). E, pairwise distance-distribution function of αN-catenin scattering data. F, comparison of the R_g and D_max values obtained for αN-catenin to values previously reported for other α-catenins and vinculin (60, 61).

FIGURE 6.

Comparison of the multidomain structures of vinculin and αN-catenin. Three individual domains (N, M, and C) of α-catenin share high structural homology with their equivalent domains of vinculin (D1, D3–D4, and D5) (supplemental Fig. S3A, S2E, and S7A). A, the elongated structure of full-length αN-catenin. B, the full-length crystal structure of vinculin (PDB code 1ST6) in the globular state. The actin-binding D5 domain forms interdomain interactions with the vinculin head region consisting of D1–D4 domains in a “pincer-shape” (56). C, superposition of αN-catenin and vinculin (gray). The D2 domain of vinculin is shown as a transparent ribbon to help visualize other structural features. The spatial arrangements of the N and M domains of α-catenin highly resemble the “pincer”-shaped arrangement of D1, D3, and D4 domains required for clamping the D5 domain and partially occluding the actin-binding sites (56).

FIGURE 7.

Model for α-catenin-mediated localization of vinculin at AJs. A, model of tension-dependent activation of α-catenin involving multistate conformational changes. α-Catenin acts as a “molecular switch” to modulate the connection between the cadherin-catenin complex and the actin cytoskeleton. In the no-tension state, the VBS within the M_I region remains closed (OFF) as there is no force being sensed by the C domain. In the high-tension state, the actomyosin-generated contractile force is detected by the C domain and helps to trigger “open to closed” conformational changes (black arrows) within the M domain. The VBS in the open (ON) conformation allows the activated vinculin to localize with the cadherin-catenin complex and engage in F-actin binding. B, model of the tension-dependent AJ-actin cytoskeleton connections regulated by cadherin-catenin-vinculin complexes.