Inhibition of SRP-dependent protein secretion by the bacterial alarmone (p)ppGpp

Abstract

The stringent response enables bacteria to respond to nutrient limitation and other stress conditions through production of the nucleotide-based second messengers ppGpp and pppGpp, collectively known as (p)ppGpp. Here, we report that (p)ppGpp inhibits the signal recognition particle (SRP)-dependent protein targeting pathway, which is essential for membrane protein biogenesis and protein secretion. More specifically, (p)ppGpp binds to the SRP GTPases Ffh and FtsY, and inhibits the formation of the SRP receptor-targeting complex, which is central for the coordinated binding of the translating ribosome to the SecYEG translocon. Cryo-EM analysis of SRP bound to translating ribosomes suggests that (p)ppGpp may induce a distinct conformational stabilization of the NG domain of Ffh and FtsY in Bacillus subtilis but not in E. coli.

Fig. 1. Post-translational targeting of SRP substrate YohP is inhibited by (p)ppGpp.

a YohP was in vitro synthesized using a purified coupled transcription/translation system (CTF system) and translation was terminated by the addition of chloramphenicol (35 mg mL⁻¹). Samples were then centrifuged for removing ribosomes and aggregates and the supernatant was incubated with INV (inner membrane vesicles) or INV-buffer for 10 min in the presence of 10 µM GTP. Where indicated, ppGpp or pppGpp were added together with INV. Subsequently, one half of the sample was directly TCA precipitated, while the other half was first treated with proteinase K (prot K) before TCA precipitation. Samples were then separated by SDS-PAGE and analyzed by autoradiography. b Quantification of three independent experiments as described in a and the mean values (±SD) are shown. c As in a, but insertion was analyzed in the presence of liposomes of reconstituted SecYEG-proteoliposomes. Liposomes were generated from E. coli phospholipids and contained 70% PE, 25% PG and 5% CL, and proteoliposomes contained 100 ng µL⁻¹ SecYEG complex. When indicated, insertion was performed in the presence of absence of purified SRP/FtsY (20 ng µL⁻¹, each) and at the indicated (p)ppGpp concentrations. Uncropped images are shown in the Supplementary Material (Supplementary Fig. 2). Samples were further processed as described in a. d Quantification of three independent experiments as described in c and the mean values (±SD) are shown.

Fig. 2. Co-translational membrane targeting of ribosome-nascent chains (RNCs) is inhibited by (p)ppGpp.

a and b RNCs of the SRP substrates FtsQ (a) or LepB (b) were in vitro synthesized (input, Inp) and incubated with INV (inner membrane vesicles) or reconstituted SecYEG proteoliposomes (SecYEG-PL; 100 ng SecYEG µL⁻¹). INV buffer and liposomes (lipos) served as a control. When indicated, purified SRP and FtsY (20 ng µL⁻¹ each) and ppGpp or pppGpp (50 µM final concentration) were present during incubation. Samples were then subjected to floatation gradient centrifugation and the membrane fraction (MF) and soluble fraction (SF) were were separated and analyzed by SDS-PAGE and autoradiography. Uncropped images are shown in the Supplementary Material (Supplementary Fig. 2) c Quantification of membrane targeting of FtsQ-RNCs (n = 3, biologically independent experiments) and LepB-RNCs (n = 4, biologically independent experiments) in the presence of (p)ppGpp. Radioactively labeled bands in the MF and SF were quantified using the ImageQuant software and are displayed as MF/(SF + MF). Error bars indicate the SEM values, which were determined using GraphPad Prism.

Fig. 3. (p)ppGpp reduces GTPase activity and complex formation of SRP and FtsY.

a Domain architecture of the bacterial SRP-GTPases Ffh (blue) and FtsY (cyan), both sharing the conserved GTPase-containing NG domain. The G-elements G1–G5 as well as the A and M domains specific to FtsY and Ffh, respectively, are shown. b Scheme of the experimental setup for analyzing the impact of increasing concentrations of (p)ppGpp on the GTPase activities of SRP and FtsY. Orange sphere depicts the signal peptide (SP), and gray strands the SRP RNA. c and d GTPase activity of full-length Ffh and FtsY-NG was assayed in the presence of increasing amounts of the competitors ppGpp (c) and pppGpp (d). Where indicated, 5 µM (Ec)FtsY-NG, 6 µM of 4.5S RNA, 5 µM Esp-signal peptide and 100 µM C₁₂E₈ (signal peptide mimic) were added to the reaction including 5 µM full-length (Ec)Ffh and 1 mM GTP. The data represent mean values (±SD) of n = 3 replicates. e The table lists the K_D values obtained for the binding of GDP, GTP, ppGpp and pppGpp to (Ec)Ffh, (Ec)FtsY- and (Bs)Ffh-NG domains determined either by isothermal titration calorimetry (ITC) or microscale thermophoresis (MST). f Scheme of the experimental setup for analyzing the impact of increasing concentrations of (p)ppGpp on the GTP-dependent formation of the Ffh/FtsY-NG domain complex. g Analytical size-exclusion chromatography (SEC) monitoring the complex formation and dissociation of Ffh-NG and FtsY-NG (100 µM each) incubated with 1 mM GMPPNP and in the absence or presence of increasing ppGpp concentrations (0, 0.5, 1, and 1.5 mM). h and i Percentage of formed Ffh-NG/FtsY-NG complexes (50 µM each) in the presence of different GMPPNP concentrations (250, 500, and 1,000 µM) analyzed in the presence of increasing ppGpp (h) and pppGpp (i) concentrations, respectively.

Fig. 4. (p)ppGpp binds in the nucleotide-binding pocket of the Ffh and FtsY-NG domains.

a Overall topology of (Ec)FtsY-NG in complex with pppGpp. b Zoom into the active site of (Ec)FtsY-NG bound to pppGpp highlighting the residues involved in ligand binding. c Overall topology of (Ec)Ffh-NG in complex with pppGpp. d Detailed view of the active site and the residues involved in binding of pppGpp. e Overall topology of the Ffh/FtsY-NG domain heterodimer from Thermus aquaticus (Ta) bound to two GCP (GppCp, a non-hydrolysable GTP-analog) molecules mimicking the binding of GTP in the twinned nucleotide-binding site (PDB-ID: 1OKK). f Zoom into the nucleotide-binding pocket shared between the NG domains of Ffh and FtsY. The 3’-OH of one GCP molecule interacts with the γ-phosphate of the opposing GCP molecule and vice versa. g Overlay of the Ffh/FtsY heterodimer with the pppGpp-bound structures of (Ec)Ffh and (Ec)FtsY (this study). Close up of the shared nucleotide-binding site shows that the δ- and ε-phosphates at the 3′-OH position of the ribose moiety of (p)ppGpp will lead to charge repulsion, where the hydrogen bond is formed in the heterodimer. Reciprocal arrangement of the two GTP nucleotides in the shared catalytic side of the Ffh/FtsY heterodimer would thereby be prevented.

Fig. 5. Cryo-EM structures of SRP-RNC complexes.

a pppGpp dataset cryo-EM map of (Bs)SRP-bound MifM-stalled RNCs filtered at local resolution, small 30S subunit in yellow and large 50S subunit in gray, SRP 6S RNA in red and Ffh in blue. SRP consists of two functional domains: the signal sequence recognition domain (S domain) and the translational elongation arrest domain (Alu domain). The S domain of the SRP is homologous to the domains II–IV of the eukaryotic 7S RNA and carries the Ffh subunit whereas the Alu domain resembles the domain I of the 7S RNA and binds to the GTPase center of the ribosome. b Molecular model of (Bs)SRP-bound MifM-stalled RNCs. c Zoomed view of the Ffh-M domain with signal sequence (SS) bound and transparent cryo-EM density in gray (right). d Comparison of Ffh-NG domain cryo-EM density for the pppGpp and GMPPNP dataset. e Zoomed view of the S domain of SRP and position of nucleotide-binding site in the Ffh-NG domain (left); comparison with previously found positions of the NG domain in bacteria (PDB 5GAF, E. coli) and eukaryotes (PDB 3JAJ, Oryctolagus cuniculus) (right). Structures are aligned on the large ribosomal subunit.

Fig. 6. Schematic summary of the bimodal interference of the alarmones (p)ppGpp with the post- and co-translational SRP-dependent membrane-targeting pathway.

In unstressed cells, SRP (Ffh in blue and SRP RNA in gray) usually recognizes a signal peptide (SP, orange) at the ribosomal exit tunnel (co-translational) but can recognize some proteins also after they have been released from the ribosome (post-translational). Binding of a GTP (green) to both SRP and the SRP receptor FtsY (light blue) then allows the formation of the SRP-FtsY-targeting complex, which leads to stimulation of GTP hydrolysis and transfer of the RNC to the SecYEG translocon (light green). In contrast, under stringent stress conditions, (p)ppGpp (red) binds to SRP and prevents formation of the SRP-FtsY-targeting complex through steric hindrance both during post- and co-translation membrane targeting.