SRP RNA controls a conformational switch regulating the SRP–SRP receptor interaction

Neher, Saskia B.; Bradshaw, Niels; Floor, Stephen N.; Gross, John D.; Walter, Peter

doi:10.1038/nsmb.1467

Cited by 40 publications

(56 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…1C). A 10-fold increased GTPase activity was previously reported for the unlinked FtsY 219 and Ffh proteins in the absence of SRP RNA (27); the smaller stimulation we observed was likely due to the presence of SRP RNA in our experiment that also activates GTP hydrolysis for the wild-type Ffh-FtsY complex.…”

Section: Resultssupporting

confidence: 64%

See 1 more Smart Citation

Ribosome–SRP–FtsY cotranslational targeting complex in the closed state

Loeffelholz

Jiang

Ariosa

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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 helices 24 and 59. The SRP-FtsY GTPases are detached from the RNA tetraloop and flexible, thus liberating the ribosomal exit site for binding of the translocation machinery.protein targeting | signal recognition particle | signal sequence | ribosome | single-particle electron cryomicroscopy T he Escherichia coli signal recognition particle (SRP) is a complex consisting of the universally conserved protein Ffh and 4.5S RNA, which adopts a hairpin structure (1). Ffh is composed of the N-terminal domain, the G domain that harbors GTPase activity, and the C-terminal methionine-rich M domain that interacts with 4.5S RNA (2, 3) and with the signal sequence (4, 5). The N and G domains form a compact structural and functional unit termed "the NG domain." Targeting of ribosome-nascent chain complexes (RNC) containing a signal sequence depends on the interaction of the RNC-SRP complex with the SRP receptor FtsY, which is membrane associated (6-9). FtsY and Ffh interact via their homologous NG domains and form a composite GTPase active site (10, 11). Crystal structures of the M domain reveal a hydrophobic groove used to capture signal sequences (4,5,12).Protein targeting is driven by highly regulated conformational rearrangements of SRP and FtsY as well as GTP hydrolysis. SRP recognizes and tightly binds to RNCs displaying a signal sequence (cargo). Next, RNC-bound SRP efficiently recruits FtsY to form a nucleotide-independent, transient early state that rearranges to a GTP-stabilized closed state (13). Ultimately, in the activated state, handover of the RNC to the Sec translocon takes place, followed by GTP hydrolysis and disassembly of the SRP-FtsY complex (14-16). These distinct conformational transitions are regulated by the ribosome and translocon in the membrane, leading to a switch from cargo recognition by SRP to cargo release (17, 18).Cryo-EM structures of bacterial SRP-bound RNCs revealed a tight cargo-recognition complex (19,20). In the SRP-FtsY early complex an overall detachment of SRP from the ribosome was observed (21). In this sta...

show abstract

Section: Resultssupporting

confidence: 64%

“…This variant lacks the N-terminal A domain and the first helix of the N domain, which was reported to inhibit FtsY-Ffh complex formation and GTPase activation (27); based on this, a single-chain construct comprising the FtsY 219 construct fused via a 31-aa glycine-and serine-rich linker to full-length Ffh (scSRP 219 ) was generated (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

Ribosome–SRP–FtsY cotranslational targeting complex in the closed state

Loeffelholz

Jiang

Ariosa

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…In the crystal structure of the SRP-FtsY GTPase heterodimer, ␣N1 of the N domain is absent in both proteins (33,34). Deletion of FtsY ␣N1 greatly facilitates SRPFtsY complex formation and enhances basal GTP hydrolysis (35), similar to what we observed in the presence of phospholipids. We hypothesized that phospholipid interaction of the MTS results in a similar conformation of ␣N1 as seen in the SRP-FtsY heterodimer.…”

Section: Mts Does Not Respond To Nucleotide-induced Conformational Chsupporting

confidence: 71%

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

Stjepanović

Kapp

Bange

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

Co-translational protein targeting to the membrane is mediated by the signal recognition particle and its receptor (FtsY). Their homologous GTPase domains interact at the membrane and form a heterodimer in which both GTPases are activated. The prerequisite for protein targeting is the interaction of FtsY with phospholipids. However, the mechanism of FtsY regulation by phospholipids remained unclear. Here we show that the N terminus of FtsY (A domain) is natively unfolded in solution and define the complete membrane-targeting sequence. We show that the membrane-targeting sequence is highly dynamic in solution, independent of nucleotides and directly responds to the density of anionic phospholipids by a random coil-helix transition. This conformational switch is essential for tethering FtsY to membranes and activates the GTPase for its subsequent interaction with the signal recognition particle. Our results underline the dynamics of lipid-protein interactions and their importance in the regulation of protein targeting and translocation across biological membranes.The biogenesis of most membrane proteins and many secretory proteins depends on the signal recognition particle (SRP) 4 and the SRP receptor (SR). SRP binds to N-terminal signal sequences of nascent polypeptide chains at the ribosome (ribosome-nascent chain complexes (RNCs)) and acts as an adaptor between the ribosome and the membrane-embedded translocation channel (1-3). Interaction of SRP with SR (FtsY in bacteria and archaea and SR␣ in eukarya) specifies the target membrane and allows for the precise coordination of RNC release from SRP and its transfer to the translocation channel. Protein targeting critically depends on the two homologous GTPases present in SRP and SR forming a heterodimer (4). GTP binding to SRP and SR is required for heterodimer formation and GTP hydrolysis triggers the dissociation of the SRP-SR complex which resets the SRP system for a new round of translocation (5). Although FtsY does not contain a hydrophobic, transmembrane sequence, it was shown to be almost exclusively localized at the membrane (6). FtsY contains three domains: an N-terminal negatively charged A domain of unknown structure and function and the highly conserved N and G domains that form a structural and functional unit, the NG domain (7, 8) (see Fig. 1A). The A domain acts as negative regulator of the FtsY GTPase in a lipid-free environment (9) and was suggested to participate in membrane interaction of FtsY by its N-terminal region (10). However, the A domain is not essential in Escherichia coli as a truncation variant (termed NGϩ1) is functional in vivo (11). It was shown that the FtsY GTPase is activated by anionic phospholipids (9) and that the membrane interaction of FtsY is crucial for the release of RNCs from the SRP-FtsY complex (12, 13), which was confirmed recently (14). Membrane interaction of E. coli FtsY depends on a conserved motif at the N terminus of the N domain, referred to as membrane-targeting sequence (MTS) (15). Recently, strong genetic ...

show abstract

“…Two conserved motifs at the N-G-domain interface, ALLEADV on the N-domain and DARGG on the G-domain, act as a fulcrum that mediates the repositioning of the N-domain relaThis article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08 -10 -0989) on July 8, 2009. tive to the G-domain in both SRP and SR (Focia et al, 2004). In addition, an inhibitory element from the first helix of the N-domain is removed (Neher et al, 2008). These structural rearrangements bring the two N-domains into proximity with one another, allowing them to make additional interface contacts that stabilize the complex (Egea et al, 2004;Focia et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

“…tive to the G-domain in both SRP and SR (Focia et al, 2004). In addition, an inhibitory element from the first helix of the N-domain is removed (Neher et al, 2008). These structural rearrangements bring the two N-domains into proximity with one another, allowing them to make additional interface contacts that stabilize the complex (Egea et al, 2004;Focia et al, 2004).…”

mentioning

confidence: 99%

A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)–SRP Receptor Interaction by the Chloroplast SRP Pathway

Jaru-Ampornpan

Nguyen

Shan

2009

MBoC

View full text Add to dashboard Cite

Cotranslational protein targeting by the signal recognition particle (SRP) requires the SRP RNA, which accelerates the interaction between the SRP and SRP receptor 200-fold. This otherwise universally conserved SRP RNA is missing in the chloroplast SRP (cpSRP) pathway. Instead, the cpSRP and cpSRP receptor (cpFtsY) by themselves can interact 200-fold faster than their bacterial homologues. Here, cross-complementation analyses revealed the molecular origin underlying their efficient interaction. We found that cpFtsY is 5-to 10-fold more efficient than Escherichia coli FtsY at interacting with the GTPase domain of SRP from both chloroplast and bacteria, suggesting that cpFtsY is preorganized into a conformation more conducive to complex formation. Furthermore, the cargo-binding M-domain of cpSRP provides an additional 100-fold acceleration for the interaction between the chloroplast GTPases, functionally mimicking the effect of the SRP RNA in the cotranslational targeting pathway. The stimulatory effect of the SRP RNA or the M-domain of cpSRP is specific to the homologous SRP receptor in each pathway. These results strongly suggest that the M-domain of SRP actively communicates with the SRP and SR GTPases and that the cytosolic and chloroplast SRP pathways have evolved distinct molecular mechanisms (RNA vs. protein) to mediate this communication. INTRODUCTIONThe signal recognition particle (SRP) and the SRP receptor (SR) comprise the major cellular machineries that cotranslationally deliver newly synthesized proteins from the cytosol to target membranes (Walter and Johnson, 1994;Keenan et al., 2001). Cotranslational protein targeting begins with recognition of the cargo-ribosomes translating nascent polypeptides containing signal sequences-by the SRP (Walter et al., 1981). The cargo is brought to the vicinity of the target membrane via the interaction between the SRP and SRP receptor (FtsY in bacteria) (Gilmore et al., 1982a). On arrival at the membrane, SRP unloads its cargo to the protein-conducting channel, composed of the sec61p complex in eukaryotic cells or secYEG complex in bacteria and archaea (Simon and Blobel, 1991;Gorlich et al., 1992;Halic et al., 2006). The SRP and SRP receptor also reciprocally stimulate each other's GTPase activity (Powers and Walter, 1995). Thus, after cargo unloading, guanosine triphosphate (GTP) hydrolysis drives disassembly of the SRP • SR complex, returning the components into the cytosol for the next round of protein targeting (Connolly et al., 1991).The SRP pathway is conserved throughout all three kingdoms of life. Although the protein components of SRP and SR vary across species, the functional core of SRP is a highly conserved ribonucleoprotein complex, composed of a 54-kDa SRP GTPase (SRP54 in eukaryotes or Ffh in bacteria) and an SRP RNA (Keenan et al., 2001). The SRP receptor also contains a conserved GTPase domain that is highly homologous to the GTPase domain in SRP54, and together the GTPase domains of SRP and SR form a unique subgroup in the GTPase superfamily (Keen...

show abstract

SRP RNA controls a conformational switch regulating the SRP–SRP receptor interaction

Cited by 40 publications

References 41 publications

Ribosome–SRP–FtsY cotranslational targeting complex in the closed state

Ribosome–SRP–FtsY cotranslational targeting complex in the closed state

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

A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)–SRP Receptor Interaction by the Chloroplast SRP Pathway

Contact Info

Product

Resources

About