The Cryogenic Electron Microscopy Structure of the Cell Adhesion Regulator Metavinculin Reveals an Isoform-Specific Kinked Helix in Its Cytoskeleton Binding Domain

Abstract

Vinculin and its heart-specific splice variant metavinculin are key regulators of cell adhesion processes. These membrane-bound cytoskeletal proteins regulate the cell shape by binding to several other proteins at cell-cell and cell-matrix junctions. Vinculin and metavinculin link integrin adhesion molecules to the filamentous actin network. Loss of both proteins prevents cell adhesion and cell spreading and reduces the formation of stress fibers, focal adhesions, or lamellipodia extensions. The binding of talin at cell-matrix junctions or of α-catenin at cell-cell junctions activates vinculin and metavinculin by releasing their autoinhibitory head-tail interaction. Once activated, vinculin and metavinculin bind F-actin via their five-helix bundle tail domains. Unlike vinculin, metavinculin has a 68-amino-acid insertion before the second α-helix of this five-helix F-actin-binding domain. Here, we present the full-length cryogenic electron microscopy structure of metavinculin that captures the dynamics of its individual domains and unveiled a hallmark structural feature, namely a kinked isoform-specific α-helix in its F-actin-binding domain. Our identified conformational landscape of metavinculin suggests a structural priming mechanism that is consistent with the cell adhesion functions of metavinculin in response to mechanical and cellular cues. Our findings expand our understanding of metavinculin function in the heart with implications for the etiologies of cardiomyopathies.

Keywords: actin; cadherin; cancer; catenin; cell adhesion; cell junction; cell migration; cell signaling; heart failure; integrin; plasma membrane.

Figure 1

Metavinculin structure. (A) Top, Metavinculin domain structure. Metavinculin is organized in four domains colored spectrally (Vh1, orange; Vh2, yellow; Vh3, green; Vt2, blue) that make up the vinculin head (VH) domain that is connected to the metavinculin tail (MVt, violet) domain. The metavinculin-specific insert between vinculin residues 915 and 916 spanning part of the linker and the MVt domain is boxed and indicated by two asterisks. Left, bottom: Vh1, Vh2, and Vh3 have each two subdomains, and their residue range is indicated below the N-term and C-term labels (for N-terminal and C-terminal subdomain, respectively). Right, bottom: MVt is a five-helix bundle domain (residue range of each α-helix is indicated) with an isoform-specific (black borders) coiled coil and first α-helix H1’. (B) Superposition of the patient-derived Δ954 metavinculin polypeptide chains in the asymmetric unit in the crystal (gray) onto the two wild type polypeptide chains in the asymmetric unit (Vh1, orange; Vh2, yellow; Vh3, green; Vt2, blue; MVt, violet). Inter-molecular interactions are indicated. (C) Superposition of the two polypeptide chains in the asymmetric unit from the structure of the patient-derived ∆954 metavinculin. Subunit A is shown in gray and subunit B is colored spectrally (Vh1, orange; Vh2, yellow; Vh3, green; Vt2, blue; MVt, violet) and its linker region that could be built only in subunit B is shown in black (residues 840–857). Relative domain differences are indicated.

Figure 2

Cryogenic electron microscopy model of full-length metavinculin. (A). Top panel, Representative aligned and motion corrected images for metavinculin particles. Bottom panel, Representative two-dimensional classes for metavinculin particles exhibiting visible helix bundle features. (B) Top panel, The 4.17 Å metavinculin structure as obtained using all particles is depicted with the docked full-length human metavinculin structure color coded spectrally (Vh1, orange; Vh2, yellow; Vh3, green; Vt2, blue; MVt, violet). Bottom panel, The gold standard Fourier shell correlation curve using a 0.143 Å threshold, as obtained from homogeneous refinement from all particles, is provided for our final 4.17 Å metavinculin structure. (C) Local resolution map to show the overall resolution coverage along the entire metavinculin structure. The scale bar is color coded from high (3.5 Å, blue) to low (10 Å, red) resolution.

Figure 3

Coordinated metavinculin domain movements confers conformational flexibility. Our metavinculin structure derived from successive heterogeneous and homogeneous refinement elucidates discrete auto-inhibited conformations of metavinculin. The refined metavinculin conformer is colored spectrally (Vh1, orange; Vh2, yellow; Vh3, green; Vt2, blue; MVt, violet). The bottom panels show the gold standard Fourier shell coefficient derived from homogeneous refinement applying a 0.143 Å threshold limit for resolution estimation. (A) The 4.15 Å resolution H1’-parallel metavinculin structure is shown with an overlay of the corresponding refined model. (B) The 4.5 Å resolution H1’-kinked metavinculin structure exhibiting differently arranged subdomains is shown with an overlay of the corresponding refined model. (C) The 4.27 Å resolution H1’-parallel metavinculin structure is shown with an overlay of the corresponding refined model.

Figure 4

Interdomain distinctions between the H1’-parallel and H1’-kinked metavinculin conformers. (A) Superposition of both Vh1 subdomains (Vh1-N for N-terminal and Vh1-C for C-terminal, residues 2–250) and tail MVt (residues 975–1114; Vt in vinculin) domains from our two vinculin subunits in the crystal, our four metavinculin subunits in the crystal, and our three cryogenic electron microscopy structures (all in gray except for the H1’-kinked metavinculin conformer where Vh1 is in orange and MVt in violet) shows that these two domains are almost identical. Cα positions 961–972 of the three cryogenic electron microscopy structures are highlighted as spheres to show the novel kink in the isoform-specific H1’ α-helix (arrow). The termini (residues 1 and 1119) are labeled as N and C, respectively. (B) Superposition of the Vh1 (residues 2–250) and MVt (residues 975–1114) domains from our H1’-kinked (with domains colored spectrally: Vh1, orange; Vh2, yellow; Vh3, green; Vt2, blue; MVt, violet) and H1’-parallel (shown in gray) metavinculin cryogenic electron microscopy structures. Cα positions 961–972 of the three cryogenic electron microscopy structures are highlighted as spheres to show the novel kink in the isoform-specific H1’ α-helix (arrow). Several subdomains are labeled, and their relative movements are indicated. (C) Superposition of the Vh1 (residues 2–250) and MVt (residues 975–1114) domains from our H1’-kinked (with domains colored spectrally) and H1’-parallel (shown in gray) metavinculin cryogenic electron microscopy structures. Several subdomains are labeled, and the relative movements of the 4 α-helices of the C-terminal Vh3 subdomain are indicated.

Figure 5

Inter-domain distinctions between the H1’-parallel metavinculin conformers. (A) Superposition of both Vh1 subdomains (Vh1-N and Vh1-C, residues 2–250) and the MVt (residues 975–1114) domain from our two H1’-parallel metavinculin cryogenic electron microscopy structures. The 4.15 Å H1’-parallel metavinculin conformer has its domains colored spectrally (Vh1, orange; Vh2, yellow; Vh3, green; Vt2, blue; MVt, violet) and the 4.27 Å H1’-parallel metavinculin conformer is shown in gray. Several subdomains are labeled, and the relative movements of the two Vh2 subdomains are indicated. (B) Superposition of the Vh1 (residues 2–250) and MVt (residues 975–1114) domains from our H1’-parallel metavinculin cryogenic electron microscopy (colored spectrally) and crystal (subunit A, shown in gray) structures. Several subdomains are labeled, and relative subdomain movements are indicated. (C) Superposition of the Vh1 (residues 2–250) and MVt (residues 975–1114) domains from our H1’-parallel metavinculin cryogenic electron microscopy (colored spectrally) and crystal (subunit B, shown in gray) structures. The crystal structure has residues through 859 for VH and 1131 for MVt, while the cryogenic electron microscopy structure has residues built through 839 for VH and 1119 (labeled) for MVt. Subunit B residues Asn-773, Glu-775, Asp-1042, and Arg-1046 are also labeled by their respective amino acid one letter codes (N, E, D, R).

Figure 6

Conformational flexibility as determined by three-dimensional variability analyses. Three-dimensional variability analyses of metavinculin particles of the primed interface in the H1’-kinked state (red) and H1’-parallel state (blue) of metavinculin depicting the overall mobility of the various subdomains. The left panel show the prominent lateral movements of the Vh1 and Vh3 C-terminal four-helix bundles relative to each other. The right panels represent the 90° rotated view and illustrates the lateral movement of the Vh2 C-terminal four-helix bundle toward the Vh1 N-terminal four-helix bundle as a consequence of coordinated movement of rest of the four-helical bundles. The middle panels show the superposed view of the observed states with the top panel showing one of the states and the bottom panel showing the other state in opposite direction. The arrows indicate the direction of movement of individual subdomains, and the solid and dashed lines distinguish the lateral movements corresponding to each other. The various domains positions are labeled for clarity.

Figure 7

Mechanism of primed interface states exhibited by metavinculin. The cartoon representation of the overall conformation, similar to our crystal structure, is as depicted in the bottom. A slight twist in the four-helix bundles of Vh2 and Vh3 leads to the second H1’-parallel conformation (top right). Either of the H1’-parallel conformations (top right or bottom) transitions to the H1’-kinked conformation accommodating very large movements in the Vh1, Vh3, Vt2, and MVt subdomains with the separation of α-helix H1’ depicted with white separator. (top left). The interplay between these states as seen in our cryogenic electron microscopy structures provides a glimpse of the primed interface state of metavinculin. The subdomains are color coded to represent Vh1 (orange), Vh2 (yellow), Vh3 (green), Vt2 (blue), and MVt (purple). The single arrows indicate dhte domain movements, the double arrows the transitions from one metavinculin conformer to another.