The Structure of the Mammalian Signal Recognition Particle (SRP) Receptor as Prototype for the Interaction of Small GTPases with Longin Domains

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 SR␣ and SR␤, which is membrane-anchored. Here we present the 2.45-Å structure of mammalian SR␤ in its Mg 2؉ GTP-bound state in complex with the minimal binding domain of SR␣ termed SRX. SR␤ 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 SR␤-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 SR␤-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 SR␤-GTP:SRX structure as a prototype for conserved interactions in a variety of GTPase regulated targeting events occurring at endomembranes. Nascent chains of secretory and membrane proteins are targeted via the ribonucleoprotein particle SRP 2 and the interaction with its receptor (SR) to the translocation machinery within the endoplasmic reticulum (ER) membrane (1, 2). The function of the SRP system is described by the SRP cycle (3, 4). SRP recognizes N-terminal hydrophobic signal sequences as soon as they emerge from the ribosomal polypeptide exit tunnel. The complex is targeted to the SR at the membrane, and the ribosome-nascent chain (RNC) complex is transferred to the translocon. Upon GTP hydrolysis in SRP and SR the complex dissociates.The eukaryotic SR consists of the two GTPases SR␣ and SR␤ (1, 5). SR␣ is a multidomain SRP GTPase with a characteristic low affinity for nucleotide (ϳ10 M) and is stable in the nucleotide-free form (6 -8). The C-terminal part of SR␣ (FtsY in archaea and eubacteria) contains the stable NG domain, which binds to the respective NG domain of SRP (SRP54, Ffh in eubacteria). Several structures of the isolated NG domains revealed the basis for the SRP GTPase cycle (for reviews, see Refs. 2-4 and 9), 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). The N-terminal part of SR␣ is responsible for tethering SR␣ to the ER membrane bound SR␤ (12). The interaction localizes to the globular SRX domain of SR␣ comprising the first 130 residues (13). SRX has been described as effector for SR␤ and only binds to the GTP-bound form of the GTPase (13,14). The SRX domain belongs to the SNARE-like superfamily including the N-terminal domains of non-syntaxin SNAREs, also known as longin domains (13, 15). Longin domains have been proposed to regulate a variety of membrane trafficki...

Lipids Trigger a Conformational Switch That Regulates Signal Recognition Particle (SRP)-mediated Protein Targeting

“…However, further experiments are needed to clarify whether the A domain interacts exclusively with lipids as suggested (10) or whether a yet undefined protein component contributes to its membrane interaction. In eukaryotes, the N terminus of the A domain (X2 domain) of SR␣ is permanently tethered to the transmembrane protein SR␤ via a longin domain interaction (47)(48)(49). Taken together, our results underline the importance of membrane lipids in the spatiotemporal restriction of protein activity to membranes, and we envision that other membrane-associated protein complexes have adapted similar mechanisms.…”

Identification of the Yeast R-SNARE Nyv1p as a Novel Longin Domain-containing Protein

“…The molecular details underlying these apparent differences between mammalian AP2 and yeast AP3 require detailed structural information on AP3. It is noteworthy that the chain and the N-terminal region of the mu chain of adaptins contain a longin fold (Collins et al, 2002;Heldwein et al, 2004) as apparently do the and ␦ subunits of the COPI coat protein complex (Schlenker et al, 2006). The significance of a shared fold in the vesicle coats and cargo sorting complexes that presumably recognize and sort longin SNAREs merits investigation.…”

Signal Recognition Particle Receptor Exposes the Ribosomal Translocon Binding Site

“…Crystal structures of the SRaX1-SRb complex (8,12) show GTP present in the active site, suggesting catalytic inactivity of SRb in complex with SRa. The interaction between SRaX and SRb is GTP dependent, and GTP hydrolysis would result in dissociation (8).…”