RESUMEN
BACKGROUND: The PDZ-LIM proteins are a family of signalling adaptors that interact with the actin cross-linking protein, alpha-actinin, via their PDZ domains or via internal regions between the PDZ and LIM domains. Three of the PDZ-LIM proteins have a conserved 26-residue ZM motif in the internal region, but the structure of the internal region is unknown. RESULTS: In this study, using circular dichroism and nuclear magnetic resonance (NMR), we showed that the ALP internal region (residues 107-273) was largely unfolded in solution, but was able to interact with the alpha-actinin rod domain in vitro, and to co-localize with alpha-actinin on stress fibres in vivo. NMR analysis revealed that the titration of ALP with the alpha-actinin rod domain induces stabilization of ALP. A synthetic peptide (residues 175-196) that contained the N-terminal half of the ZM motif was found to interact directly with the alpha-actinin rod domain in surface plasmon resonance (SPR) measurements. Short deletions at or before the ZM motif abrogated the localization of ALP to actin stress fibres. CONCLUSION: The internal region of ALP appeared to be largely unstructured but functional. The ZM motif defined part of the interaction surface between ALP and the alpha-actinin rod domain.
Asunto(s)
Actinina/química , Proteínas de Microfilamentos/química , Actinina/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Línea Celular Tumoral , Colorantes Fluorescentes/química , Humanos , Proteínas con Dominio LIM , Proteínas de Microfilamentos/biosíntesis , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Earlier reports have shown that ALP has an internal interaction site. We were able to stabilize the structure of this unfolded part to a great extent by aspartic acid, which allowed the backbone assignment. No secondary structure of the polypeptide was observed.