Abstract
The eukaryotic signal recognition particle (SRP) and its receptor (SR) play a central role in co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum. The SR is a heterodimeric complex assembled by the two GTPases SRalpha and SRbeta, which is membrane-anchored. Here we present the 2.45-A structure of mammalian SRbeta in its Mg2+ GTP-bound state in complex with the minimal binding domain of SRalpha termed SRX. SRbeta is a member of the Ras-GTPase superfamily closely related to Arf and Sar1, while SRX belongs to the SNARE-like superfamily with a fold also known as longin domain. SRX binds to the P loop and the switch regions of SRbeta-GTP. The binding mode and structural similarity with other GTPase-effector complexes suggests a co-GAP (GTPase-activating protein) function for SRX. Comparison with the homologous yeast structure and other longin domains reveals a conserved adjustable hydrophobic surface within SRX which is of central importance for the SRbeta-GTP:SRX interface. A helix swap in SRX results in the formation of a dimer in the crystal structure. Based on structural conservation we present the SRbeta-GTP:SRX structure as a prototype for conserved interactions in a variety of GTPase regulated targeting events occurring at endomembranes.
Highlights
The eukaryotic SR consists of the two GTPases SR␣ and SR [1, 5]
Several structures of the isolated NG domains revealed the basis for the signal recognition particle (SRP) GTPase cycle, and the complex of the two NG domains shows a remarkably symmetric heterodimer with the nucleotides in direct contact at the center of the interface [10, 11]
We analyze the fundamentals of the longin domain family and suggest the interaction of small GTPases and longin domains to be important for targeting of large complexes or vesicles to the endomembrane system
Summary
Protein Expression and Purification—The His6-tagged N-terminal 176 amino acids from human SR␣ (including SRX, residues 1–130) together with mouse SR lacking the N-terminal transmembrane region (here referred to as SR, residues 58 –269) were expressed as a bi-cistronic construct from vector pET16b (Stratagene) in BL21(DE3) Escherichia coli cells (Stratagene). The protein was purified via affinity tag purification using Ni2ϩ-loaded chelating Sepharose Fast Flow beads (Amersham Biosciences). The protein was further purified via ion exchange chromatography (Q- and SP-Sepharose) and via size exclusion chromatography (Superdex 200, Amersham Biosciences) using a low salt buffer (10 mM Tris/HCl pH 8.0, 150 mM NaCl, 5 mM MgCl2, and 1 mM dithiothreitol). Crystals grew within 4 – 6 weeks over a reservoir containing 100 mM sodium citrate, pH 5.5, 2.0 M (NH4)2SO4, and in the presence of 100 mM guanidinium chloride. The asymmetric unit contains one SR-GTP:SRX heterodimer corresponding to a solvent content of 55% and a Matthews coefficient of 2.7 Å3/Da. Crystals were flash-frozen in liquid nitrogen using 20% (v/v) glycerol as cryo-protectant. Diffraction data were measured at the European Synchroton Radiation Facility in Grenoble, France at beamline ID14 – 4.
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