RESUMEN
Here, we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously, we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct filamentous actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and cross-linking. Sedimentation equilibrium assays indicate that the Ig3 domain of palladin does not self-associate. These combined data are consistent with an actin cross-linking mechanism that involves concurrent attachment of two actin filaments by a single palladin molecule by an electrostatic mechanism. Palladin mutations that disrupt actin binding show altered cellular distributions and morphology of actin in cells, revealing a functional requirement for the interaction between palladin and actin in vivo.
Asunto(s)
Actinas/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/fisiología , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiología , Dominios y Motivos de Interacción de Proteínas , Actinas/química , Sustitución de Aminoácidos/fisiología , Animales , Células COS , Chlorocebus aethiops , Proteínas del Citoesqueleto/genética , Inmunoglobulinas/química , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosfoproteínas/genética , Unión Proteica/genética , Dominios y Motivos de Interacción de Proteínas/genética , Mapeo de Interacción de Proteínas , Transporte de Proteínas/genética , Conejos , TransfecciónRESUMEN
Palladin is a recently described phosphoprotein that plays an important role in cell adhesion and motility. Previous studies have shown that palladin overexpression results in profound changes in actin organization in cultured cells. Palladin binds to the actin-associated proteins alpha-actinin, vasodilator-stimulated phosphoprotein, profilin, Eps8, and ezrin, suggesting that it may affect actin organization indirectly. To determine its molecular function in generating actin arrays, we purified palladin and asked if it is also capable of binding to F-actin directly. In co-sedimentation and differential sedimentation assays, palladin was found to both bind and cross-link actin filaments. This bundling activity was confirmed by fluorescence and electron microscopy. Palladin fragments were then purified and used to determine the sequences necessary to bind and bundle F-actin. The Ig3 domain of palladin bound to F-actin, and a palladin fragment containing Ig3, Ig4, and the region linking these domains was identified as a fragment that was able to bundle F-actin. Because palladin has multiple Ig domains, and only one of them binds to F-actin, this suggests that different Ig domains may be specialized for distinct biological functions. In addition, our results suggest a potential role for palladin in generating specialized, actin-based cell morphologies via both direct actin cross-linking activity and indirect scaffolding activity.
Asunto(s)
Citoesqueleto de Actina/química , Proteínas del Citoesqueleto/química , Fosfoproteínas/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Animales , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/aislamiento & purificación , Proteínas del Citoesqueleto/metabolismo , Humanos , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Microscopía Electrónica de Transmisión , Fosfoproteínas/genética , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de FluorescenciaRESUMEN
As part of our NMR structure determination of the palladin Ig3 domain, we report nearly complete NMR chemical shift assignments for the (1)H, (13)C, and (15)N nuclei.
Asunto(s)
Proteínas del Citoesqueleto/química , Espectroscopía de Resonancia Magnética/métodos , Fosfoproteínas/química , Secuencia de Aminoácidos , Isótopos de Carbono/química , Datos de Secuencia Molecular , Peso Molecular , Isótopos de Nitrógeno/química , Estructura Terciaria de Proteína , ProtonesRESUMEN
Mounting evidence suggests that the focal adhesion targeting (FAT) domain, an antiparallel four-helix bundle, exists in alternative conformations that may modulate phosphorylation, ligand binding, and the subcellular localization of focal adhesion kinase (FAK). In order to characterize the conformational dynamics of the FAT domain, we have developed a novel method for reconstructing the folding pathway of the FAT domain by using discrete molecular dynamics (DMD) simulations, with free energy constraints derived from NMR hydrogen exchange data. The DMD simulations detect a folding intermediate, in which a cooperative unfolding event causes helix 1 to lose helical character while separating from the helix bundle. The conformational dynamic features of helix 1 in the intermediate state of the FAT domain are likely to facilitate Y926 phosphorylation, yet interfere with paxillin binding. The presence of this intermediate state in vivo may promote FAK signaling via the ERK/MAPK pathway and by release of FAK from focal adhesions.
