Structural basis of signal sequence surveillance and selection by the SRP–FtsY complex (original) (raw)

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...

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.

Targeting proteins to membranes: structure of the signal recognition particle

In all three kingdoms of life, co-translational targeting of secretory and membrane proteins to the prokaryotic plasma membrane or eukaryotic endoplasmic reticulum is mediated by a ribonucleoprotein complex, the signal recognition particle (SRP), and its membrane-associated receptor (SR). SRP binds to signal sequences of nascent proteins as they emerge from the exit tunnel of the ribosome. The resulting targeting complex, composed of the SRP and the ribosome-nascent chain complex (RNC), then docks with the SR in a GTPdependent manner. Passing through a complex series of conformational states, SRP and SR deliver the RNC to the translocon, which in turn mediates protein translocation across or integration into the membrane. The core structural and mechanistic principles of SRP-dependent protein targeting are universally conserved. Recent structural investigations combining X-ray crystallography and cryo-electron microscopy have provided new insights into three essentials steps of the SRP-dependent protein targeting cycle: the assembly and interaction of the SRP ribonucleoprotein core, the GTP-dependent SRP-SR association, and the interaction between SRP and the ribosome. Addresses

Construction of the ‘minimal’ SRP that interacts with the translating ribosome but not with specific membrane receptors in Escherichia coli

FEBS Letters, 2002

Escherichia coli signal recognition particle (SRP) consists of 4.5S RNA and Ffh protein. In contrast to eukaryotes, it remains unclear whether translation arrest takes place in prokaryotic cells. To study this problem we constructed a fusion of the M domain of Ffh protein with a cleavable affinity tag. This mutant Ffh, in a complex with 4.5S RNA, can bind signal peptide at the translating ribosome but is unable to bind the membrane. This SRP^ribosome complex should accumulate in the cell if translation is arrested. To test this, the complex was purified from the cells by ultracentrifugation and affinity chromatography. The composition of the complex was analyzed and found to consist of ribosomal RNAs and proteins, the Ffh M domain and 4.5S RNA. The accumulation of this complex in the cell in significant amounts indicated that SRP-mediated translation arrest did occur in bacterial cells.

Predominant membrane localization is an essential feature of the bacterial signal recognition particle receptor

BMC Biology, 2009

Background: The signal recognition particle (SRP) receptor plays a vital role in co-translational protein targeting, because it connects the soluble SRP-ribosome-nascent chain complex (SRP-RNCs) to the membrane bound Sec translocon. The eukaryotic SRP receptor (SR) is a heterodimeric protein complex, consisting of two unrelated GTPases. The SRβ subunit is an integral membrane protein, which tethers the SRP-interacting SRα subunit permanently to the endoplasmic reticulum membrane. The prokaryotic SR lacks the SRβ subunit and consists of only the SRα homologue FtsY. Strikingly, although FtsY requires membrane contact for functionality, cell fractionation studies have localized FtsY predominantly to the cytosolic fraction of Escherichia coli. So far, the exact function of the soluble SR in E. coli is unknown, but it has been suggested that, in contrast to eukaryotes, the prokaryotic SR might bind SRP-RNCs already in the cytosol and only then initiates membrane targeting.

Cotranslational protein targeting to the membrane: Nascent-chain transfer in a quaternary complex formed at the translocon

Scientific Reports

Membrane proteins in bacteria are cotranslationally inserted into the plasma membrane through the SecYEG translocon. Ribosomes exposing the signal-anchor sequence (SAS) of a membrane protein are targeted to the translocon by the signal recognition particle (SRP) pathway. SRP scans translating ribosomes and forms high-affinity targeting complexes with those exposing a SAS. Recognition of the SAS activates SRP for binding to its receptor, FtsY, which, in turn, is primed for SRP binding by complex formation with SecYEG, resulting in a quaternary targeting complex. Here we examine the effect of SecYEG docking to ribosome-nascent-chain complexes (RNCs) on SRP binding and SAS transfer, using SecYEG embedded in phospholipid-containing nanodiscs and monitoring FRET between fluorescencelabeled constituents of the targeting complex. SecYEG-FtsY binding to RNC-SRP complexes lowers the affinity of SRP to both ribosome and FtsY, indicating a general weakening of the complex due to partial binding competition near the ribosomal peptide exit. The rearrangement of the quaternary targeting complex to the pre-transfer complex requires an at least partially exposed SAS. The presence of SecYEG-bound FtsY and the length of the nascent chain strongly influence nascent-chain transfer from SRP to the translocon and repositioning of SRP in the post-transfer complex. Extracytosolic proteins constitute about one third of the bacterial proteome and there are various pathways to target these proteins to the membrane in order to insert them into the lipid bilayer or translocate them through the membrane. In gram-negative bacteria, most inner-membrane proteins are cotranslationally targeted to the SecYEG translocon via the signal recognition particle (SRP) pathway. SRP rapidly scans ribosomes by forming transient complexes. On binding to a ribosome-nascent-chain complex (RNC) exposing the signal-anchor sequence (SAS) of a membrane protein, SRP undergoes a conformational change that results in the formation of a high-affinity complex 1-6. The formation of the complex of SRP and RNC protects the SAS from other interaction partners such as ribosome-associated chaperones and nascent-chain modifying enzymes 7-10. Additionally, binding to RNCs activates SRP by engaging the C-terminal M domain of SRP protein Ffh in SAS binding and the N domain in interaction with ribosomal protein uL23. In this way the NG (GTPase) domain becomes available for binding to the homologous NG domain of the SRP receptor, FtsY 11-15. Similarly, the free form of FtsY is in an inactive"closed" state where the N-terminal, membrane-binding A domain and the C-terminal NG domain are engaged in a strong interaction with one another 16. On binding to the translocon in the membrane, FtsY is activated by an engagement of the A domain with SecYEG and membrane phospholipids 16,17. This frees the NG domain of FtsY for the interaction with the homologous NG domain of SRP protein Ffh 18. These activation events lead to an increased affinity of SRP binding to FtsY and take place at the ribosome or at SecYEG 1,2,16,19,20. In addition, the interaction between the SecYEG translocon and the ribosome is enhanced in the presence of the nascent chain of a membrane protein 21,22. The increased affinity between SRP and FtsY and between RNC and SecYEG favors the assembly of the quaternary targeting/transfer complex at the translocon 21,22. However, a detailed description of the events taking place at the membrane is still lacking. Structural and biochemical data suggested that SRP and SecYEG interact with the ribosome in a mutually exclusive

The signal recognition particle binds to protein L23 at the peptide exit of the Escherichia coli ribosome

RNA, 2003

The signal recognition particle (SRP) from Escherichia coli, composed of Ffh protein and 4.5S RNA, mediates membrane targeting of translating ribosomes displaying a signal or signal-anchor sequence. SRP binds at the peptide exit of the large ribosomal subunit. Structural details of the interaction are not known. Here, the position of Ffh or SRP on the ribosome was probed by using site-specific UV-induced crosslinking by p-azidophenacyl bromide (AzP) attached to a number of cysteine residues engineered into surface positions of Ffh. Efficient crosslinking to vacant ribosomes took place from two positions (AzP17 and AzP25) in the N domain of Ffh, both with Ffh and SRP. Both AzP17 and AzP25 were predominantly crosslinked to ribosomal protein L23 that is located at the peptide exit of the 50S subunit. The SRP receptor, FtsY, did not change the crosslink pattern, whereas the presence of a nascent signal peptide on the ribosome resulted in a second crosslink between Ffh(AzP17) and protein L23, indicating that binding to the nascent signal peptide induced a slightly different arrangement of SRP on the ribosome. These results indicate a model of the topographical arrangement of SRP at the peptide exit of the 50S ribosomal subunit.

Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

Molecular Biology of the Cell, 1998

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound ...