Structural Characterisation of the Mammalian SRP Receptor

Abstract

In eukaryotes, secretory and membrane proteins are targeted to the endoplasmic reticulum (ER) membrane for cotranslational translocation. This requires the specific interaction of the signal recognition particle (SRP), a ribonucleoprotein, with its receptor (SR). The eukaryotic SR is a heterodimeric protein consisting of SRα and SRβ which is anchored to the ER membrane. In all three kingdoms of life the conserved GTPases in SRP and SR (in eukaryotes SRP54 and SRα, respectively) enable the formation of the docking complex in a GTP dependent manner. SRβ is the third and least understood GTPase participating in cotranslational targeting in eukaryotes. Therefore a more detailed view on the GTPase cycle of SRβ and functionally relevant SRβ effector interactions should be obtained. X-ray structure analysis was used to determine the structure of a soluble form of SRβ (SRβΔTM) in its GTP bound state in complex with a fragment of SRα. The structure allows to precisely define the minimal SRβ binding domain of SRα (SRX). The homology to other small GTPases and the underlying principles of regulation together with previous biochemical data allow to attribute a functional role to SRX in the activation of the SRβ GTPase. An immobilised peptide library was used in order to examine the interaction of SR with the translocon. Evidence is presented that the apo- and likely the GDP bound form of SRβΔTM (SRβΔTM-apo/GDP) bind to cytosolic loops of the translocon in contrast to SRβΔTM-GTP in complex with SRXHis or SRαHis. These experiments suggest that the SRX binding surface of SRβ-GTP, as observed in the SRXHis:βΔTMGTP X-ray structure, is the same as engaged in SRβΔTM-apo/GDP binding to the translocon. SRβΔTM-apo binds to peptides of several cytosolic loop regions of the translocon. Mapping of these regions on the available structure of the homologous translocon from Methanococcus jannaschii suggests that SRβΔTM-apo/GDP blocks the translocation pore. Based on the molecular structure of the SRXHis:βΔTM-GTP SRX belongs to the SNARE-like superfamily with the common fold of the longin domains (LDs). The common principles of the LD family are analysed by a comparison of surface hydropathicity and structure based sequence alignment with structurally known LDs. Putative LDs are considered according to secondary structure prediction and primary sequence alignment. The interaction of small GTPases with LDs is suggested to be important for the assembly of large complexes at or targeting of vesicles to the endomembrane system. Important structural information on the mammalian SRP:SR complex are still missing, including the arrangement of the individual protein subunits and the positioning of the SRP RNA. Extensive purification and assembly of a pentameric SRP:SR complex, consisting of SRα:βΔTM, SRP54, SRP19 and a 104 base pair long SRP RNA, was set up in order to establish the basis for further structural studies on this macromolecular complex.