RESUMO
Vinculin, a scaffolding protein that localizes to focal adhesions (FAs) and adherens junctions, links the actin cytoskeleton to the adhesive super-structure. While vinculin binds to a number of cytoskeletal proteins, it can also associate with phosphatidylinositol 4,5-bisphosphate (PIP2) to drive membrane association. To generate a structural model for PIP2-dependent interaction of vinculin with the lipid bilayer, we conducted lipid-association, nuclear magnetic resonance, and computational modeling experiments. We find that two basic patches on the vinculin tail drive membrane association: the basic collar specifically recognizes PIP2, while the basic ladder drives association with the lipid bilayer. Vinculin mutants with defects in PIP2-dependent liposome association were then expressed in vinculin knockout murine embryonic fibroblasts. Results from these analyses indicate that PIP2 binding is not required for localization of vinculin to FAs or FA strengthening, but is required for vinculin activation and turnover at FAs to promote its association with the force transduction FA nanodomain.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Adesões Focais/metabolismo , Bicamadas Lipídicas/química , Fosfatidilinositol 4,5-Difosfato/química , Vinculina/química , Citoesqueleto de Actina/genética , Actinas/genética , Motivos de Aminoácidos , Animais , Sítios de Ligação , Embrião de Mamíferos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Adesões Focais/ultraestrutura , Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/metabolismo , Mecanotransdução Celular , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica , Vinculina/genética , Vinculina/metabolismoRESUMO
Vinculin is an essential adhesion protein that links membrane-bound integrin and cadherin receptors through their intracellular binding partners to filamentous actin, facilitating mechanotransduction. Here we present an 8.5-Å-resolution cryo-electron microscopy reconstruction and pseudo-atomic model of the vinculin tail (Vt) domain bound to F-actin. Upon actin engagement, the N-terminal "strap" and helix 1 are displaced from the Vt helical bundle to mediate actin bundling. We find that an analogous conformational change also occurs in the H1' helix of the tail domain of metavinculin (MVt) upon actin binding, a muscle-specific splice isoform that suppresses actin bundling by Vt. These data support a model in which metavinculin tunes the actin bundling activity of vinculin in a tissue-specific manner, providing a mechanistic framework for understanding metavinculin mutations associated with hereditary cardiomyopathies.