Structures of SRP54 and SRP19, the Two Proteins that Organize the Ribonucleic Core of the Signal Recognition Particle from Pyrococcus furiosus Pascal F. Egea 1 *, Johanna Napetschnig 2 , Peter Walter 1 , Robert M. Stroud 1 * 1 Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, United States of America, 2 Laboratory of Cell Biology and Howard Hughes Medical Institute, The Rockefeller University, New York, New York, United States of America Abstract In all organisms the Signal Recognition Particle (SRP), binds to signal sequences of proteins destined for secretion or membrane insertion as they emerge from translating ribosomes. In Archaea and Eucarya, the conserved ribonucleoproteic core is composed of two proteins, the accessory protein SRP19, the essential GTPase SRP54, and an evolutionarily conserved and essential SRP RNA. Through the GTP-dependent interaction between the SRP and its cognate receptor SR, ribosomes harboring nascent polypeptidic chains destined for secretion are dynamically transferred to the protein translocation apparatus at the membrane. We present here high-resolution X-ray structures of SRP54 and SRP19, the two RNA binding components forming the core of the signal recognition particle from the hyper-thermophilic archaeon Pyrococcus furiosus (Pfu). The 2.5 A ˚ resolution structure of free Pfu-SRP54 is the first showing the complete domain organization of a GDP bound full-length SRP54 subunit. In its ras-like GTPase domain, GDP is found tightly associated with the protein. The flexible linker that separates the GTPase core from the hydrophobic signal sequence binding M domain, adopts a purely a-helical structure and acts as an articulated arm allowing the M domain to explore multiple regions as it scans for signal peptides as they emerge from the ribosomal tunnel. This linker is structurally coupled to the GTPase catalytic site and likely to propagate conformational changes occurring in the M domain through the SRP RNA upon signal sequence binding. Two different 1.8 A ˚ resolution crystal structures of free Pfu-SRP19 reveal a compact, rigid and well-folded protein even in absence of its obligate SRP RNA partner. Comparison with other SRP19NSRP RNA structures suggests the rearrangement of a disordered loop upon binding with the RNA through a reciprocal induced-fit mechanism and supports the idea that SRP19 acts as a molecular scaffold and a chaperone, assisting the SRP RNA in adopting the conformation required for its optimal interaction with the essential subunit SRP54, and proper assembly of a functional SRP. Citation: Egea PF, Napetschnig J, Walter P, Stroud RM (2008) Structures of SRP54 and SRP19, the Two Proteins that Organize the Ribonucleic Core of the Signal Recognition Particle from Pyrococcus furiosus. PLoS ONE 3(10): e3528. doi:10.1371/journal.pone.0003528 Editor: Shuguang Zhang, Massachusetts Institute of Technology, United States of America Received September 10, 2008; Accepted October 3, 2008; Published October 27, 2008 Copyright: ß 2008 Egea et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH grant GM60641 to R.M.S. that supports P.F.E and NIH grants to P.W. P.W is an investigator from the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pascal@msg.ucsf.edu (PFE); stroud@msg.ucsf.edu (RMS) Introduction In all living cells the signal recognition particle (SRP) recognizes nascent polypeptides destined for secretion or membrane insertion as they emerge from translating ribosomes [1,2]. As SRP binds to signal sequences emerging from the ribosomes, the resulting complex composed of the SRP and the ribosome-nascent chain complex is then targeted towards the membrane through the GTP-dependent interaction with the membrane-associated SRP receptor (SR also named FtsY in bacteria). Both SRP and SR contain GTPase domains. Their tight association triggers the reciprocal activation of GTP hydrolyses that govern docking and release of the ribosome-nascent chain to the translocon and recycling of the SRP. Although the SRP pathway is evolutionarily conserved, the composition of the SRP and its receptor SR varies widely. All SRPs from bacteria to eukaryotes and archaea, with the exception of chloroplastic SRPs, require the essential SRP RNA to function. SRP RNA has been shown to play a central role in the protein targeting reaction by catalyzing the interaction between SRP and its receptor [3] but also, albeit to a lesser extent, in accelerating GTP hydrolysis in the SRPNSR complex once formed [4]. In eukaryotes, the SRP contains six proteins (SRP68/SRP72, SRP9/SRP14, SRP54 and SRP19) and a 300-nucleotide RNA. Most bacterial systems display a simplest organization with a shorter RNA (about 113 nucleotides) and a single protein subunit Ffh, the homologue of SRP54. Archeal SRPs contain an SRP RNA of similar size and fold to that in eukaryotes but only two proteins, SRP54 and SRP19 (Figure 1A). Thus, archaeal SRPs represent a more streamlined version of the eukaryotic homo- logues and provide an opportunity to explore an increased repertoire in structural and biophysical terms. Although the overall composition of the SRP systems differ, the central ribonucleoprotein core and the general mechanism of GTP-dependent targeting are highly conserved. Since SRP54 is the only protein subunit conserved in all SRPs, it represents the key component in protein targeting. It is essential for signal sequence recognition and binding at the ribosome, and for the GTP-dependent interaction with SR its cognate receptor. This interaction determines proper transfer of the ribosome-nascent PLoS ONE | www.plosone.org 1 October 2008 | Volume 3 | Issue 10 | e3528