Structural basis of signal sequence surveillance and selection by the SRP-FtsY complex

Abstract

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.

Figure 1. The N-terminal extension of EspP inhibits co-translational protein targeting but does not affect RNC-SRP binding

(a) Signal sequences of EspP and its variants used in this study. The N-terminal extension of the signal sequence and the signal peptide cleavage site are indicated. The classical signal sequence is marked by a red box, and mutations are highlighted by red letters. (b)In vitro targeting and translocation efficiency of EspP signal sequence variants fused to Prolactin using microsomal membranes. (c) Equilibrium titration of RNC-SRP binding. Error bars represent standard deviation, with n = 3..

Figure 2. The EspPN-terminal extension leads to a weaker and distorted early targeting complex

(a) Equilibrium titration of the SRP•FtsY early complex in the presence of RNCs bearing EspP signal sequence variants. 300–500 nM RNCs were used to ensure that most of the SRP is ribosome-bound. Error bars represent standard deviation, with n = 3. (b) Cryo-EM structure of RNC^EspP-SRP-FtsY shown with the view into the polypeptide exit tunnel. The large ribosomal subunit (50S) is depicted in blue, the small ribosomal subunit (30S) in yellow and the single chain SRP-FtsY in red. (c,d) EM reconstruction and quasi-atomic model of the RNC^EspP-SRP-FtsY early complex (c) in a close-up view from the back of the 50S subunit and (d) in a view as in (b). The experimental density is shown in light grey, 4.5S RNA in orange, the EspP signal sequence (ss) in red, the Ffh M-domain in yellow, the Ffh NG-domain in greenyellow, the FtsY NG-domain in magenta, the 50S rRNA in dark gray, ribosomal proteins L24 in purple and L22 in skyblue. The density of the ribosome is not shown in (d) for clarity.

Figure 3. M-domain arrangement during co-translational targeting

(a) Close-up view on the exit of the ribosomal tunnel and the M-domain of the RNC^FtsQ-SRP cryo-EM structure. Color coding is the same as in Figure 2c. (b) Same view as in (a) for the RNC^FtsQ-SRP-FtsY early complex. (c) Cryo-EM structure of the RNC^EspP-SRP-FtsY early complex shown in the same view as (a,b) for comparison. (d) Close-up on the M-domain with the signal sequence in the EM reconstruction and quasi-atomic model of the RNC^EspP-SRP-FtsY complex. Color coding is the same as in Figure 2d,e. The positions of the Ffh M- and G-domains are indicated.

Figure 4. Ffh-FtsY NG-domain arrangement in the ‘false’ early complex formed with EspP compared to the productive early complex formed with a correct cargo

(a) In the RNC^EspP•SRP•FtsY ‘false’ early complex, the Ffh-FtsY NG-domains have a weak interface involving the N-domain of Ffh and the NG-domain of FtsY. The experimental density is depicted in pale cyan; unfilled density indicates flexibility in this part. (b) Pseudo-symmetric V-shaped NG-domain arrangement in the early targeting complex with RNC^{FtsQ 20}. The experimental density of this complex is shown in light pink. (c,d) Overlays of (c) the EM densities of the early targeting complexes from (a) & (b), and (d) the corresponding quasi-atomic models. Arrows indicate positional differences of the NG-domains. For the overlays (c,d), the RNA tetraloops of the structures (c) and models (d) have been aligned. Color codings of the quasi-atomic models are as in Figure 2 except in (d), where the NG-domain complex of the early targeting complex with RNC^FtsQ is depicted in grey for clarity.

Figure 5. The N-terminal extension leads to a less productive early complex and slower assembly of the closed SRP-FtsY complex

(a) Kinetics for rearrangement of the early to the closed complex for EspP and variants. (b) Assembly rates of the closed SRP-FtsY complex mediated by RNCs displaying EspP signal sequence variants. Error bars represent standard deviation, with n = 4.

Figure 6. Model of signal sequence surveillance by the SRP and FtsY

The SRP tightly binds to correct cargos. FtsY binding leads to detachment of SRP from the ribosome, and a pseudo-symmetric NG-domain arrangement. EspP has a less hydrophobic signal sequence, leading to a moderate affinity of SRP (13 nM). FtsY has a lower affinity (311 nM) for this SRP-RNC^EspP complex and forms a less favourable, distorted and flexible Ffh-FtsY NG-domain heterodimer. The EspP ‘false’ early targeting complex NG-domains that are loosely associated and rearrange inefficiently into the closed/activated state leading to premature FtsY dissociation rather than successful completion of the targeting reaction.