RESUMEN
Eph receptor tyrosine kinases and their ephrin ligands regulate cell navigation during normal and oncogenic development. Signaling of Ephs is initiated in a multistep process leading to the assembly of higher-order signaling clusters that set off bidirectional signaling in interacting cells. However, the structural and mechanistic details of this assembly remained undefined. Here we present high-resolution structures of the complete EphA2 ectodomain and complexes with ephrin-A1 and A5 as the base unit of an Eph cluster. The structures reveal an elongated architecture with novel Eph/Eph interactions, both within and outside of the Eph ligand-binding domain, that suggest the molecular mechanism underlying Eph/ephrin clustering. Structure-function analysis, by using site-directed mutagenesis and cell-based signaling assays, confirms the importance of the identified oligomerization interfaces for Eph clustering.
Asunto(s)
Receptor EphA1/química , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Efrina-A1/química , Efrina-A1/genética , Efrina-A1/metabolismo , Efrina-A5/química , Efrina-A5/genética , Efrina-A5/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptor EphA1/genética , Receptor EphA1/metabolismo , Receptor EphA2/química , Receptor EphA2/genética , Receptor EphA2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
The majority of current high-throughput protein purification protocols include rate-limiting centrifugation steps. A column and nozzle assembly was developed that can be used in-line with microfluidization for the purification of bacterially-overexpressed, His-tagged proteins directly from bacterial cultures. Yields and purity are comparable with standard protocols. This large-scale protein purification protocol is easy to use and widely-applicable.