RNA-mediated interaction between the peptide-binding and GTPase domains of the signal recognition particle (original) (raw)
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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 Å 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 Å 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.
Proceedings of the National Academy of Sciences, 2007
The signal-recognition particle (SRP) is a ubiquitous protein-RNA complex that targets proteins to cellular membranes for insertion or secretion. A key player in SRP-mediated protein targeting is the evolutionarily conserved core consisting of the SRP RNA and the multidomain protein SRP54. Communication between the SRP54 domains is critical for SRP function, where signal sequence binding at the M domain directs receptor binding at the GTPase domain (NG domain). These SRP activities are linked to domain rearrangements, for which the role of SRP RNA is not clear. In free SRP, a direct interaction of the GTPase domain with SRP RNA has been proposed but has never been structurally verified. In this study, we present the crystal structure at 2.5-Å resolution of the SRP54-SRP19-SRP RNA complex of Methanococcus jannaschii SRP. The structure reveals an RNA-bound conformation of the SRP54 GTPase domain, in which the domain is spatially well separated from the signal peptide binding site. The association of both the N and G domains with SRP RNA in free SRP provides further structural evidence for the pivotal role of SRP RNA in the regulation of the SRP54 activity.
Structural basis for the molecular evolution of SRP-GTPase activation by protein
Nature Structural & Molecular Biology, 2011
a r t i c l e s SIMIBI-class (named after the signal recognition particle, MinD, BioD) nucleotide-binding proteins appeared early in evolution 1 and contain GTPases, as well as ATPases, involved in the correct localization of cellular constituents. The MinD ATPase, as the central part of the Min system, regulates the determination of the cell division site in all bacterial species 2 . SRP-GTPases form a subfamily of the SIMIBI class, with only three members: the signal sequence-binding protein Ffh (SRP54 in Eukarya and Archaea), the SRP receptor FtsY (SRα in Eukarya) and FlhF, which is involved in flagella biosynthesis 3-5 . They share the conserved NG domain, which contains two major additions to the conserved fold of small G proteins. First, an α-β-α element (I-box) is inserted in the effector region; second, the N domain, comprising four α-helices, is attached to the N terminus of the G domain. SRP (Ffh together with the SRP RNA) and FtsY constitute the universally conserved co-translational protein-targeting machinery 6,7 . When bound to GTP, Ffh and FtsY form, through interactions between their NG domains 8,9 , a heterodimeric complex that regulates the transfer of a ribosome-nascent chain complex to a vacant translocon in the membrane with a series of conformational rearrangements 10,11 . The two GTPases share a composite active site between their G domains in which GTP hydrolysis is reciprocally activated 12 . The SRP RNA 13-15 and membrane lipids 16,17 play fundamental roles in activating the Ffh-FtsY GTPases. The recent structure of the SRP-FtsY complex, together with biochemical implications, suggest that the distal end of the hairpin-like SRP RNA may be involved in this activation 18 . The third SRP-GTPase FlhF, together with the MinD-type protein YlxH (also known as FlhG, FleN, motR or MinD2), is essential for the placement and assembly of flagella 19 in many polar and peritrichous flagellated bacteria 20-24 . FlhF is required for the targeting of the first flagellar protein, FliF, to the cell pole 25 by a mechanism that is so far poorly understood. FlhF is associated with the membrane 25,26 and localizes at the cell pole 20 . The FlhF protein contains an N-terminal B domain that seems to be involved in FliF targeting 25 ; it shares the NG domain fold with the other two members of the SRP-GTPase subfamily. FlhF forms a stable homodimer with GTP and a composite active site that is basically identical to the active site of the Ffh-FtsY heterodimer 5 . In both the homo-and heterodimer, the two nucleotides are bound in a head-to-tail manner, with the γ-phosphate of one nucleotide interacting with the 3′-OH of the ribose moiety of the other. However, for the homo-and heterodimers formed by the three SRP-GTPases, the molecular mechanism of activation is still unknown. We set out to understand the activation of SRP-GTPases by studying FlhF.
Activated GTPase movement on an RNA scaffold drives co-translational protein targeting
Approximately one-third of the proteome is initially destined for the eukaryotic endoplasmic reticulum or the bacterial plasma membrane 1 . The proper localization of these proteins is mediated by a universally conserved protein-targeting machinery, the signal recognition particle (SRP), which recognizes ribosomes carrying signal sequences 2-4 and, through interactions with the SRP receptor 5,6 , delivers them to the protein-translocation machinery on the target membrane 7 . The SRP is an ancient ribonucleoprotein particle containing an essential, elongated SRP RNA for which precise functions have remained elusive. Here we used single-molecule fluorescence microscopy to show that the Escherichia coli SRP-SRP receptor GTPase complex, after initial assembly at the tetraloop end of SRP RNA, travels over 100 Å to the distal end of this RNA, where rapid GTP hydrolysis occurs. This movement is negatively regulated by the translating ribosome and, at a later stage, positively regulated by the SecYEG translocon, providing an attractive mechanism for ensuring the productive exchange of the targeting and translocation machineries at the ribosome exit site with high spatial and temporal accuracy. Our results show that large RNAs can act as molecular scaffolds that enable the easy exchange of distinct factors and precise timing of molecular events in a complex cellular process; this concept may be extended to similar phenomena in other ribonucleoprotein complexes.
Structure of the E. coli signal recognition particle bound to a translating ribosome
Nature, 2007
The prokaryotic signal recognition particle (SRP) targets membrane proteins into the inner membrane 1-4 . It binds translating ribosomes and screens the emerging nascent chain for a hydrophobic signal sequence, such as the transmembrane helix of inner membrane proteins. If such a sequence emerges, the SRP binds tightly, allowing the SRP receptor to lock on. This assembly delivers the ribosome-nascent chain complex to the protein translocation machinery in the membrane. Using cryo-electron microscopy and single-particle reconstruction, we obtained a 16 Å structure of the Escherichia coli SRP in complex with a translating E. coli ribosome containing a nascent chain with a transmembrane helix anchor. We also obtained structural information on the SRP bound to an empty E. coli ribosome. The latter might share characteristics with a scanning SRP complex, whereas the former represents the next step: the targeting complex ready for receptor binding. High-resolution structures of the bacterial ribosome and of the bacterial SRP components are available, and their fitting explains our electron microscopic density. The structures reveal the regions that are involved in complex formation, provide insight into the conformation of the SRP on the ribosome and indicate the conformational changes that accompany high-affinity SRP binding to ribosome nascent chain complexes upon recognition of the signal sequence.
Structural insights into SRP RNA: An induced fit mechanism for SRP assembly
RNA, 2005
Proper assembly of large protein-RNA complexes requires sequential binding of the proteins to the RNA. The signal recognition particle (SRP) is a multiprotein-RNA complex responsible for the cotranslational targeting of proteins to biological membranes. Here we describe the crystal structure at 2.6-Å resolution of the S-domain of SRP RNA from the archeon Methanococcus jannaschii. Comparison of this structure with the SRP19-bound form reveals the nature of the SRP19-induced conformational changes, which promote subsequent SRP54 attachment. These structural changes are initiated at the SRP19 binding site and transmitted through helix 6 to looped-out adenosines, which form tertiary RNA interaction with helix 8. Displacement of these adenosines enforces a conformational change of the asymmetric loop structure in helix 8. In free RNA, the three unpaired bases A195, C196, and C197 are directed toward the helical axis, whereas upon SRP19 binding the loop backbone inverts and the bases are splayed out in a conformation that resembles the SRP54-bound form. Nucleotides adjacent to the bulged nucleotides seem to be particularly important in the regulation of this loop transition. Binding of SRP19 to 7S RNA reveals an elegant mechanism of how protein-induced changes are directed through an RNA molecule and may relate to those regulating the assembly of other RNPs.
Structural basis of signal sequence surveillance and selection by the SRP–FtsY complex
Nature Structural & Molecular Biology, 2013
Signal-recognition particle (SRP)-dependent targeting of translating ribosomes to membranes is a multistep quality-control process. Ribosomes that are translating weakly hydrophobic signal sequences can be rejected from the targeting reaction even after they are bound to the SRP. Here we show that the early complex, formed by Escherichia coli SRP and its receptor FtsY with ribosomes translating the incorrect cargo EspP, is unstable and rearranges inefficiently into subsequent conformational states, such that FtsY dissociation is favored over successful targeting. The N-terminal extension of EspP is responsible for these defects in the early targeting complex. The cryo-electron microscopy structure of this 'false' early complex with EspP revealed an ordered M domain of SRP protein Ffh making two ribosomal contacts, and the NG domains of Ffh and FtsY forming a distorted, flexible heterodimer. Our results provide a structural basis for SRP-mediated signal-sequence selection during recruitment of the SRP receptor.
Nature Structural & Molecular Biology, 2004
Cotranslational targeting directly couples synthesis of proteins to their translocation across or insertion into membranes. The signal recognition particle (SRP) and its membrane-bound receptor facilitate the targeting of the translation machinery, the ribosome, via recognition of a signal sequence in the nascent peptide chain. By combining structures of free and ribosome-bound SRP we derive a structural model describing the dynamic nature of SRP when it meets the ribosome.
Ribosome-SRP-FtsY cotranslational targeting complex in the closed state
Proceedings of the National Academy of Sciences of the United States of America, 2015
The signal recognition particle (SRP)-dependent pathway is essential for correct targeting of proteins to the membrane and subsequent insertion in the membrane or secretion. In Escherichia coli, the SRP and its receptor FtsY bind to ribosome-nascent chain complexes with signal sequences and undergo a series of distinct conformational changes, which ensures accurate timing and fidelity of protein targeting. Initial recruitment of the SRP receptor FtsY to the SRP-RNC complex results in GTP-independent binding of the SRP-FtsY GTPases at the SRP RNA tetraloop. In the presence of GTP, a closed state is adopted by the SRP-FtsY complex. The cryo-EM structure of the closed state reveals an ordered SRP RNA and SRP M domain with a signal sequence-bound. Van der Waals interactions between the finger loop and ribosomal protein L24 lead to a constricted signal sequence-binding pocket possibly preventing premature release of the signal sequence. Conserved M-domain residues contact ribosomal RNA h...