PPIase domain of trigger factor acts as auxiliary chaperone site to assist the folding of protein substrates bound to the crevice of trigger factor

Trigger factor (TF) is the first chaperone encountered by nascent chains in bacteria, which consists of two modules: peptidyl-prolyl-cis/trans-isomerase (PPIase) domain and a crevice built by both N- and C-terminal domains. While the crevice is suggested to provide a protective space over the peptide exit site of ribosome for nascent polypeptides to fold, it remains unclear whether PPIase domain is directly involved in assisting protein folding. Here, we introduced structural change into different regions of TF, and investigated their influence on the chaperone function of TF in assisting the folding of various substrate proteins, including oligomeric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and monomeric carbonic anhydrase II (CA II) and lysozyme. Results showed that structural disturbances by site-specific mutations in the PPIase active site or by deletion of the PPIase domain from TF affected the chaperone activity of TF toward CA II and GAPDH but had no effect on TF-assisted lysozyme refolding, suggesting PPIase domain is involved in assisting the folding of substrates larger than lysozyme. Mutants with the structural disturbances in the crevice totally lost the chaperone activity toward all the substrates we used in this investigation. These results provide further evidence to confirm that the crevice is the major chaperone site of TF, and the hydrophobic pocket in PPIase domain acts as an auxiliary site to assist the folding of substrate proteins bound to the crevice in a substrate-dependent manner, which is beneficial for TF to provide appropriate assistance for protein folding by changing protective space and binding affinity.     

Versatility of Trigger Factor Interactions with Ribosome-Nascent Chain Complexes

Trigger factor (TF) is the first molecular chaperone that interacts with nascent chains emerging from bacterial ribosomes. TF is a modular protein, consisting of an N-terminal ribosome binding domain, a PPIase domain, and a C-terminal domain, all of which participate in polypeptide binding. To directly monitor the interactions of TF with nascent polypeptide chains, TF variants were site-specifically labeled with an environmentally sensitive NBD fluorophore. We found a marked increase in TF-NBD fluorescence during translation of firefly luciferase (Luc) chains, which expose substantial regions of hydrophobicity, but not with nascent chains lacking extensive hydrophobic segments. TF remained associated with Luc nascent chains for 111 ± 7 s, much longer than it remained bound to the ribosomes (t_½ ∼ 10–14 s). Thus, multiple TF molecules can bind per nascent chain during translation. The Escherichia coli cytosolic proteome was classified into predicted weak and strong interactors for TF, based on the occurrence of continuous hydrophobic segments in the primary sequence. The residence time of TF on the nascent chain generally correlated with the presence of hydrophobic regions and the capacity of nascent chains to bury hydrophobicity. Interestingly, TF bound the signal sequence of a secretory protein, pOmpA, but not the hydrophobic signal anchor sequence of the inner membrane protein FtsQ. On the other hand, proteins lacking linear hydrophobic segments also recruited TF, suggesting that TF can recognize hydrophobic surface features discontinuous in sequence. Moreover, TF retained significant affinity for the folded domain of the positively charged, ribosomal protein S7, indicative of an alternative mode of TF action. Thus, unlike other chaperones, TF appears to employ multiple mechanisms to interact with a wide range of substrate proteins.     

Identification of a potential hydrophobic peptide binding site in the C-terminal arm of trigger factor

Trigger factor (TF) is the first chaperone to interact with nascent chains and facilitate their folding in bacteria. Escherichia coli TF is 432 residues in length and contains three domains with distinct structural and functional properties. The N-terminal domain of TF is important for ribosome binding, and the M-domain carries the PPIase activity. However, the function of the C-terminal domain remains unclear, and the residues or regions directly involved in substrate binding have not yet been identified. Here, a hydrophobic probe, bis-ANS, was used to characterize potential substrate-binding regions. Results showed that bis-ANS binds TF with a 1:1 stoichiometry and a K(d) of 16 microM, and it can be covalently incorporated into TF by UV-light irradiation. A single bis-ANS-labeled peptide was obtained by tryptic digestion and identified by MALDI-TOF mass spectrometry as Asn391-Lys392. In silico docking analysis identified a single potential binding site for bis-ANS on the TF molecule, which is adjacent to this dipeptide and lies in the pocket formed by the C-terminal arms. The bis-ANS-labeled TF completely lost the ability to assist GAPDH or lysozyme refolding and showed increased protection toward cleavage by alpha-chymotrypsin, suggesting blocking of hydrophobic residues. The C-terminal truncation mutant TF389 also showed no chaperone activity and could not bind bis-ANS. These results suggest that bis-ANS binding may mimic binding of a substrate peptide and that the C-terminal region of TF plays an important role in hydrophobic binding and chaperone function.     

Trigger factor lacking the PPIase domain can enhance the folding of eukaryotic multi-domain proteins in Escherichia coli

Recombinant expression of eukaryotic proteins in bacteria often results in misfolding and aggregation. The ribosome-binding Trigger factor (TF) is the first molecular chaperone that interacts with nascent polypeptide chains in bacteria. Here we show that mutant TF lacking the PPIase domain (TFNC) is more efficient than wild-type TF in enhancing the folding yield of multi-domain proteins such as firefly luciferase. We find that TFNC has a shorter residence time on nascent chains, thus facilitating co-translational folding. By delaying folding relative to translation, the PPIase domain may increase the propensity of misfolding for certain eukaryotic proteins that rely on a mechanism of co-translational, domain-wise folding.     

A conserved motif is prerequisite for the interaction of NAC with ribosomal protein L23 and nascent chains

In eukaryotes, newly synthesized proteins interact co-translationally with a multitude of different ribosome-bound factors and chaperones including the conserved heterodimeric nascent polypeptide-associated complex (NAC) and a Hsp40/70-based chaperone system. These factors are thought to play an important role in protein folding and targeting, yet their specific ribosomal localizations, which are prerequisite for their functions, remain elusive. This study describes the ribosomal localization of NAC and the molecular details by which NAC is able to contact the ribosome and gain access to nascent polypeptides. We identified a conserved RRK(X)nKK ribosome binding motif within the beta-subunit of NAC that is essential for the entire NAC complex to attach to ribosomes and allow for its interaction with nascent polypeptide chains. The motif localizes within a potential loop region between two predicted alpha-helices in the N terminus of betaNAC. This N-terminal betaNAC ribosome-binding domain was completely portable and sufficient to target an otherwise cytosolic protein to the ribosome. NAC modified with a UV-activatable cross-linker within its ribosome binding motif specifically cross-linked to L23 ribosomal protein family members at the exit site of the ribosome, providing the first evidence of NAC-L23 interaction in the context of the ribosome. Mutations of L23 reduced NAC ribosome binding in vivo and in vitro, whereas other eukaryotic ribosome-associated factors such as the Hsp70/40 chaperones Ssb or Zuotin were unaffected. We conclude that NAC employs a conserved ribosome binding domain to position itself on the L23 ribosomal protein adjacent to the nascent polypeptide exit site.     

Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome

As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro-synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain.     

Cotranslational protein folding within the ribosome tunnel influences trigger-factor recruitment

In recent years, various folding zones within the ribosome tunnel have been identified and explored through x-ray, cryo-electron microscopy (cryo-EM), and molecular biology studies. Here, we generated ribosome-bound nascent polypeptide complexes (RNCs) with different polyalanine (poly-A) inserts or signal peptides from membrane/secretory proteins to explore the influence of nascent chain compaction in the Escherichia coli ribosome tunnel on chaperone recruitment. By employing time-resolved fluorescence resonance energy transfer and immunoblotting, we were able to show that the poly-A inserts embedded in the passage tunnel can form a compacted structure (presumably helix) and reduce the recruitment of Trigger Factor (TF) when the helical motif is located in the region near the tunnel exit. Similar experiments on nascent chains containing signal sequences that may form compacted structural motifs within the ribosome tunnel and lure the signal recognition particle (SRP) to the ribosome, provided additional evidence that short, compacted nascent chains interfere with TF binding. These findings shed light on the possible controlling mechanism of nascent chains within the tunnel that leads to chaperone recruitment, as well as the function of L23, the ribosomal protein that serves as docking sites for both TF and SRP, in cotranslational protein targeting.     

Trigger factor binding to ribosomes with nascent peptide chains of varying lengths and sequences

Trigger factor (TF) is the first protein-folding chaperone to interact with a nascent peptide chain as it emerges from the ribosome. Here, we have used a spin down assay to estimate the affinities for the binding of TF to ribosome nascent chain complexes (RNCs) with peptides of varying lengths and sequences. An in vitro system for protein synthesis assembled from purified Escherichia coli components was used to produce RNCs stalled on truncated mRNAs. The affinity of TF to RNCs exposing RNA polymerase sequences increased with the length of the nascent peptides. TF bound to RNA polymerase RNCs with significantly higher affinity than to inner membrane protein leader peptidase and bacterioopsin RNCs. The latter two RNCs are substrates for signal recognition particle, suggesting complementary affinities of TF and signal recognition particle to nascent peptides targeted for cytoplasm and membrane.     

Three-state equilibrium of Escherichia coli trigger factor

Trigger Factor (TF) is the first chaperone that interacts with nascent chains of cytosolic proteins in Escherichia coli. Although its chaperone activity requires association with ribosomes, TF is present in vivo in a 2-3 fold molar excess over ribosomes and a fraction of it is not ribosome-associated after cell lysis. Here we show that TF follows a three-state equilibrium. Size exclusion chromatography, crosslinking and analytical ultracentrifugation revealed that uncomplexed TF dimerizes with an apparent Kd of 18 microM. Dimerization is mediated by the N-terminal ribosome binding domain and the C-terminal domain of TF, whereas the central peptidyl prolyl isomerase (PPlase) and substrate binding domain does not contribute to dimerization. Crosslinking experiments showed that TF is monomeric in its ribosome-associated state. Quantitative analysis of TF binding to ribosomes revealed a dissociation constant for the TF-ribosome complex of approximately 1.2 microM. From these data we estimate that in vivo most of the ribosomes are in complex with monomeric TF. Uncomplexed TF, however, is in a monomer-dimer equilibrium with approximately two thirds of TF existing in a dimeric state.     

Localization of the trigger factor binding site on the ribosomal 50S subunit

In Escherichia coli, protein folding is undertaken by three distinct sets of chaperones, the DnaK-DnaJ and GroEL-GroES systems and the trigger factor (TF). TF has been proposed to be the first chaperone to interact with the nascent polypeptide chain as it emerges from the tunnel of the 70S ribosome and thus probably plays an important role in co-translational protein folding. We have made complexes with deuterated ribosomes (50S subunits and 70S ribosomes) and protated TF and determined the TF binding site on the respective complexes using the neutron scattering technique of spin-contrast variation. Our data suggest that the TF binds in the form of a homodimer. On both the 50S subunit and the 70S ribosome, the TF position is in proximity to the tunnel exit site, near ribosomal proteins L23 and L29, located on the back of the 50S subunit. The positions deviate from one another, such that the position on the 70S ribosome is located slightly further from the tunnel than that determined for the 50S subunit alone. Nevertheless, from both determined positions interaction between TF and a short nascent chain of 57 amino acid residues would be plausible, compatible with a role for TF participation in co-translational protein folding.     

Ligand crowding at a nascent signal sequence

We have systematically analyzed the molecular environment of the signal sequence of a growing secretory protein from Escherichia coli using a stage- and site-specific cross-linking approach. Immediately after emerging from the ribosome, the signal sequence of pOmpA is accessible to Ffh, the protein component of the bacterial signal recognition particle, and to SecA, but it remains attached to the surface of the ribosome via protein L23. These contacts are lost upon further growth of the nascent chain, which brings the signal sequence into sole proximity to the chaperone Trigger factor (TF). In its absence, nascent pOmpA shows extended contacts with L23, and even long chains interact in these conditions proficiently with Ffh. Our results suggest that upon emergence from the ribosome, the signal sequence of an E. coli secretory protein gradually becomes sequestered by TF. Although TF thereby might control the accessibility of pOmpA's signal sequence to Ffh and SecA, it does not influence interaction of pOmpA with SecB.     

Dynamics of trigger factor interaction with translating ribosomes

In all organisms ribosome-associated chaperones assist early steps of protein folding. To elucidate the mechanism of their action, we determined the kinetics of individual steps of the ribosome binding/release cycle of bacterial trigger factor (TF), using fluorescently labeled chaperone and ribosome-nascent chain complexes. Both the association and dissociation rates of TF-ribosome complexes are modulated by nascent chains, whereby their length, sequence, and folding status are influencing parameters. However, the effect of the folding status is modest, indicating that TF can bind small globular domains and accommodate them within its substrate binding cavity. In general, the presence of a nascent chain causes an up to 9-fold increase in the rate of TF association, which provides a kinetic explanation for the observed ability of TF to efficiently compete with other cytosolic chaperones for binding to nascent chains. Furthermore, a subset of longer nascent polypeptides promotes the stabilization of TF-ribosome complexes, which increases the half-life of these complexes from 15 to 50 s. Nascent chains thus regulate their folding environment generated by ribosome-associated chaperones.     

Flexibility of the Bacterial Chaperone Trigger Factor in Microsecond-Timescale Molecular Dynamics Simulations

The bacterial chaperone trigger factor (TF) is the first chaperone to be encountered by a nascent protein chain as it emerges from the ribosome exit tunnel. Experimental results suggest that TF possesses considerable conformational flexibility, and in an attempt to provide an atomic-level view of this flexibility, we have performed independent 1.5-μs molecular dynamics simulations of TF in explicit solvent using two different simulation force fields (OPLS-AA/L and AMBER ff99SB-ILDN). Both simulations indicate that TF possesses tremendous flexibility, with huge excursions from the crystallographic conformation caused by reorientations of the protein’s constituent domains; both simulations also predict the formation of extensive contacts between TF’s PPIase domain and the Arm 1 domain that is involved in nascent-chain binding. In the OPLS simulation, however, TF rapidly settles into a very compact conformation that persists for at least 1 μs, whereas in the AMBER simulation, it remains highly dynamic; additional simulations in which the two force fields were swapped suggest that these differences are at least partly attributable to sampling issues. The simulation results provide potential rationalizations of a number of experimental observations regarding TF’s conformational behavior and have implications for using simulations to model TF’s function on translating ribosomes.     

Flexibility of the Bacterial Chaperone Trigger Factor in Microsecond-Timescale Molecular Dynamics Simulations

The bacterial chaperone trigger factor (TF) is the first chaperone to be encountered by a nascent protein chain as it emerges from the ribosome exit tunnel. Experimental results suggest that TF possesses considerable conformational flexibility, and in an attempt to provide an atomic-level view of this flexibility, we have performed independent 1.5-μs molecular dynamics simulations of TF in explicit solvent using two different simulation force fields (OPLS-AA/L and AMBER ff99SB-ILDN). Both simulations indicate that TF possesses tremendous flexibility, with huge excursions from the crystallographic conformation caused by reorientations of the protein’s constituent domains; both simulations also predict the formation of extensive contacts between TF’s PPIase domain and the Arm 1 domain that is involved in nascent-chain binding. In the OPLS simulation, however, TF rapidly settles into a very compact conformation that persists for at least 1 μs, whereas in the AMBER simulation, it remains highly dynamic; additional simulations in which the two force fields were swapped suggest that these differences are at least partly attributable to sampling issues. The simulation results provide potential rationalizations of a number of experimental observations regarding TF’s conformational behavior and have implications for using simulations to model TF’s function on translating ribosomes.     

1H, 15N and 13C assignments of the dimeric ribosome binding domain of trigger factor from Escherichia coli

Trigger factor (TF) is a multi-domain molecular chaperone that binds to the bacterial ribosome at the tunnel exit from which nascent polypeptides emerge. We present here the NMR assignments of the ribosome binding domain (RBD) of TF from Escherichia coli as a stable 26 kDa dimer, using conditions that are similar to a crystallographic study from which an X-ray crystal structure of the identical construct was determined.     

Chaperone binding at the ribosomal exit tunnel

The exit tunnel region of the ribosome is well established as a focal point for interaction between the components that guide the fate of nascent polypeptides. One of these, the chaperone trigger factor (TF), associates with the 50S ribosomal subunit through its N-terminal domain. Targeting of TF to ribosomes is crucial to achieve its remarkable efficiency in protein folding. A similar tight coupling to translation is found in signal recognition particle (SRP)-dependent protein translocation. Here, we report crystal structures of the E. coli TF ribosome binding domain. TF is structurally related to the Hsp33 chaperone but has a prominent ribosome anchor located as a tip of the molecule. This tip includes the previously established unique TF signature motif. Comparison reveals that this feature is not found in SRP structures. We identify a conserved helical kink as a hallmark of the TF structure that is most likely critical to ensure ribosome association.     

Exploring the capacity of trigger factor to function as a shield for ribosome bound polypeptide chains

Ribosome-bound trigger factor (TF) is the first chaperone encountered by a nascent polypeptide chain in bacteria. TF has been proposed to form a cradle-shaped shield for nascent chains up to approximately 130 residues to fold in a protected environment upon exit from the ribosome. We report that nascent chains of luciferase up to 280 residues in length are relatively protected by TF against digestion by proteinase K. In contrast, nascent chains of the constitutively unstructured protein alpha-synuclein were not protected, although they were in close proximity to TF by crosslinking. Thus, TF is not a general shield for nascent chains. Protease protection appears to depend on a hydrophobic interaction of TF with nascent polypeptides.     

Functional dissection of Escherichia coli trigger factor: unraveling the function of individual domains

In Escherichia coli, the ribosome-associated chaperone Trigger Factor (TF) promotes the folding of newly synthesized cytosolic proteins. TF is composed of three domains: an N-terminal domain (N), which mediates ribosome binding; a central domain (P), which has peptidyl-prolyl cis/trans isomerase activity and is involved in substrate binding in vitro; and a C-terminal domain (C) with unknown function. We investigated the contributions of individual domains (N, P, and C) or domain combinations (NP, PC, and NC) to the chaperone activity of TF in vivo and in vitro. All fragments comprising the N domain (N, NP, NC) complemented the synthetic lethality of Deltatig DeltadnaK in cells lacking TF and DnaK, prevented protein aggregation in these cells, and cross-linked to nascent polypeptides in vitro. However, DeltatigDeltadnaK cells expressing the N domain alone grew more slowly and showed less viability than DeltatigDeltadnaK cells synthesizing either NP, NC, or full-length TF, indicating beneficial contributions of the P and C domains to TF's chaperone activity. In an in vitro system with purified components, none of the TF fragments assisted the refolding of denatured d-glyceraldehyde-3-phosphate dehydrogenase in a manner comparable to that of wild-type TF, suggesting that the observed chaperone activity of TF fragments in vivo is dependent on their localization at the ribosome. These results indicate that the N domain, in addition to its function to promote binding to the ribosome, has a chaperone activity per se and is sufficient to substitute for TF in vivo.     

Cryo-EM Structures Reveal Relocalization of MetAP in the Presence of Other Protein Biogenesis Factors at the Ribosomal Tunnel Exit

During protein biosynthesis in bacteria, one of the earliest events that a nascent polypeptide chain goes through is the co-translational enzymatic processing. The event includes two enzymatic pathways: deformylation of the N-terminal methionine by the enzyme peptide deformylase (PDF), followed by methionine excision catalyzed by methionine aminopeptidase (MetAP). During the enzymatic processing, the emerging nascent protein likely remains shielded by the ribosome-associated chaperone trigger factor. The ribosome tunnel exit serves as a stage for recruiting proteins involved in maturation processes of the nascent chain. Co-translational processing of nascent chains is a critical step for subsequent folding and functioning of mature proteins.Here, we present cryo-electron microscopy structures of Escherichia coli (E. coli) ribosome in complex with the nascent chain processing proteins. The structures reveal overlapping binding sites for PDF and MetAP when they bind individually at the tunnel exit site, where L22–L32 protein region provides primary anchoring sites for both proteins. In the absence of PDF, trigger factor can access ribosomal tunnel exit when MetAP occupies its primary binding site. Interestingly, however, in the presence of PDF, when MetAP's primary binding site is already engaged, MetAP has a remarkable ability to occupy an alternative binding site adjacent to PDF. Our study, thus, discloses an unexpected mechanism that MetAP adopts for context-specific ribosome association.