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

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The Cryogenic Electron Microscopy Structure of the Cell Adhesion Regulator Metavinculin Reveals an Isoform-Specific Kinked Helix in Its Cytoskeleton Binding Domain International Journal of Molecular Sciences Article The Cryogenic Electron Microscopy Structure of the Cell Adhesion Regulator Metavinculin Reveals an Isoform-Specific Kinked Helix in Its Cytoskeleton Binding Domain Erumbi S. Rangarajan and Tina Izard * Cell Adhesion Laboratory, Department of Integrative Structural and Computational Biology, The Scripps Research Institute, Jupiter, FL 33458, USA; [email protected] * Correspondence: [email protected] 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 Citation: Rangarajan, E.S.; Izard, T. our understanding of metavinculin function in the heart with implications for the etiologies of The Cryogenic Electron Microscopy cardiomyopathies. Structure of the Cell Adhesion Regulator Metavinculin Reveals an Keywords: actin; cadherin; cancer; catenin; cell adhesion; cell junction; cell migration; cell signaling; Isoform-Specific Kinked Helix in Its heart failure; integrin; plasma membrane Cytoskeleton Binding Domain. Int. J. Mol. Sci. 2021, 22, 645. https://doi.org/10.3390/ijms22020645 1. Introduction Received: 16 December 2020 Accepted: 8 January 2021 Vinculin and its larger and muscle-specific spliced isoform called metavinculin play Published: 11 January 2021 pivotal roles in cell adhesion by regulating cellular morphology, cellular motility, and by the transducing force between neighboring cells as well as between cells and the Publisher’s Note: MDPI stays neu- extracellular matrix [1–7] At cell–cell and cell–matrix junctions, (meta)vinculin functions as tral with regard to jurisdictional clai- a scaffold where it connects transmembrane cell surface receptors to the filamentous actin ms in published maps and institutio- cytoskeleton [8–10]. nal affiliations. Vinculin is essential for the development of the heart as well as cardiomyocyte ad- hesion functions including contraction [11,12]. Vinculin knockout mouse embryos have neural tube and cardiac developmental defects and do not survive past embryonic day 10. Vinculin-null murine embryonic fibroblasts have fewer adhesions and thus have defects Copyright: © 2021 by the authors. Li- in cell spreading and are less able to attach. Therefore, they are more motile and resist censee MDPI, Basel, Switzerland. apoptosis and anoikis [13]. Moreover, heterozygous vinculin deletion (+/-) results in dilated This article is an open access article cardiomyopathies [14]. distributed under the terms and con- ditions of the Creative Commons At- Vinculin and metavinculin have seven four-helix bundle domains that are connected tribution (CC BY) license (https:// via a about 40 residue (in vinculin) or about 100 residue (in metavinculin) flexible proline- creativecommons.org/licenses/by/ rich linker to their respective and distinct five-helix bundle tail domains, which is called Vt 4.0/). in vinculin, for vinculin tail, or MVt in metavinculin, for metavinculin tail (Figure1)[ 15–18]. Int. J. Mol. Sci. 2021, 22, 645. https://doi.org/10.3390/ijms22020645 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 18 Int. J. Mol. Sci. 2021, 22, 645 2 of 19 Vt in vinculin, for vinculin tail, or MVt in metavinculin, for metavinculin tail (Figure 1) [15–18]. Metavinculin has 68 amino acids inserted into the vinculin polypeptide chain be- Metavinculintween vinculin has residues 68 amino 915 acidsand 916. inserted Structur intoally, the these vinculin additional polypeptide 68 residues chain replace between the vinculinN-terminal residues extended 915 andcoil 916.and the Structurally, first α-helix these H1 additional of the five-helix 68 residues Vt bundle replace domain, the N- terminalwhereby extended the Vt α coil-helix and H1 the in first MVtα-helix becomes H1 of part the five-helixof the head–tail Vt bundle disordered domain, wherebylinker in themetavinculin Vt α-helix H1instead. in MVt The becomes Vt and partMVt of domains the head–tail bind disorderedto F-actin, and linker their in metavinculinprimary and instead.tertiary Thestructural Vt and differences MVt domains correlate bind to with F-actin, their and distinct their primary actin bundling and tertiary and structural binding differencesproperties [19–23]. correlate with their distinct actin bundling and binding properties [19–23]. FigureFigure 1.1. MetavinculinMetavinculin structure. structure. ( A(A)) Top, Top, Metavinculin Metavinculin domain domain structure. structure. Metavinculin Metavinculin is organizedis orga- innized four in domains four domains colored colored spectrally spectrally (Vh1, orange;(Vh1, orange; Vh2, yellow; Vh2, yellow; Vh3, green;Vh3, green; Vt2, blue) Vt2, thatblue) make that up themake vinculin up the head vinculin (VH) head domain (VH) that domain is connected that is connected to the metavinculin to the metavinculin tail (MVt, violet)tail (MVt, domain. violet) The domain. The metavinculin-specific insert between vinculin residues 915 and 916 spanning part of metavinculin-specific insert between vinculin residues 915 and 916 spanning part of the linker and the linker and the MVt domain is boxed and indicated by two asterisks. Left, bottom: Vh1, Vh2, the MVt domain is boxed and indicated by two asterisks. Left, bottom: Vh1, Vh2, and Vh3 have and Vh3 have each two subdomains, and their residue range is indicated below the N-term and C- eachterm twolabels subdomains, (for N-terminal and their and C-terminal residue range subdomai is indicatedn, respectively). below the N-term Right, andbottom: C-term MVt labels is a five- (for N-terminalhelix bundle and domain C-terminal (residue subdomain, range of each respectively). α-helix is indicated) Right, bottom: with an MVt isoform-specific is a five-helix (black bundle domainborders) (residue coiled coil range and of first each α-helixα-helix H1’. is indicated)(B) Superposition with an of isoform-specific the patient-derived (black Δ borders)954 metavincu- coiled coillin polypeptide and first α-helix chains H1’. in (theB) Superpositionasymmetric unit of in the the patient-derived crystal (gray) ontoD954 the metavinculin two wild type polypeptide polypep- chainstide chains in the in asymmetric the asymmetric unit inunit the (Vh1, crystal orange; (gray) Vh2, onto yellow; the two Vh3, wild green; type polypeptide Vt2, blue; MVt, chains violet). in the asymmetricInter-molecular unit interactions (Vh1, orange; are Vh2,indicated. yellow; (C) Vh3, Superposition green; Vt2, of blue; the two MVt, polypeptide violet). Inter-molecular chains in the interactionsasymmetric areunit indicated. from the stru (C) Superpositioncture of the patient-derived of the two polypeptide ∆954 metavinculin. chains in Subunit the asymmetric A is shown unit in gray and subunit B is colored spectrally (Vh1, orange; Vh2, yellow; Vh3, green; Vt2, blue; MVt, from the structure of the patient-derived D954 metavinculin. Subunit A is shown in gray and subunit violet) and its linker region that could be built only in subunit B is shown in black (residues 840– B is colored spectrally (Vh1, orange; Vh2, yellow; Vh3, green; Vt2, blue; MVt, violet) and its linker 857). Relative domain differences are indicated. region that could be built only in subunit B is shown in black (residues 840–857). Relative domain differences are indicated. Int. J. Mol. Sci. 2021, 22, 645 3 of 19 Vinculin and metavinculin are exclusively helical, which provides these proteins with a large degree of flexibility to easily act as adaptor proteins. Two conformations have been recognized as functionally relevant: (i) their closed and inactive and (ii) their open and active states. Vinculin and metavinculin are held in their closed auto-inhibited conformers by an extensive nanomolar affinity interface between their N-terminal four-helix bundle Vh1 subdomains and their respective Vt or MVt tail domains. In turn, the vinculin and metavinculin binding partners often need to be activated first prior to binding to vinculin and metavinculin. Such activation often occurs by mechanical actomyosin force [4,24–28]. Apart from talin and actin, over a dozen other proteins also bind to vinculin and metavinculin [29]. The binding sites that have been characterized are grouped as binders to (i) the N-terminal Vh1 bundles, as seen for α-actinin [10,30–32], α-catenin [33–36], and β-catenin
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