Single‐molecule dynamics of the molecular chaperone trigger factor in living cells

In bacteria, trigger factor (TF) is the molecular chaperone that interacts with the ribosome to assist the folding of nascent polypeptides. Studies in vitro have provided insights into the function and mechanism of TF. Much is to be elucidated, however, about how TF functions in vivo. Here, we use single‐molecule tracking, in combination with genetic manipulations, to study the dynamics and function of TF in living E. coli cells. We find that TF, besides interacting with the 70S ribosome, may also bind to ribosomal subunits and form TF‐polypeptide complexes that may include DnaK/DnaJ proteins. The TF‐70S ribosome interactions are highly dynamic inside cells, with an average residence time of ～0.2 s. Our results confirm that the signal recognition particle weakens TF's interaction with the 70S ribosome, and further identify that this weakening mainly results from a change in TF's binding to the 70S ribosome, rather than its unbinding. Moreover, using photoconvertible bimolecular fluorescence complementation, we selectively probe TF₂ dimers in the cell and show that TF₂ does not bind to the 70S ribosome but is involved in the post‐translational interactions with polypeptides. These findings contribute to the fundamental understanding of molecular chaperones in assisting protein folding in living cells.     

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.     

Identification of nascent chain interaction sites on trigger factor

The role of ribosome-binding molecular chaperones in protein folding is not yet well understood. Trigger factor (TF) is the first chaperone to interact with nascent polypeptides as they emerge from the bacterial ribosome. It binds to the ribosome as a monomer but forms dimers in free solution. Based on recent crystal structures, TF has an elongated shape, with the peptidyl-prolyl-cis/trans-isomerase (PPIase) domain and the N-terminal ribosome binding domain positioned at opposite ends of the molecule and the C-terminal domain, which forms two arms, positioned in between. By using site specifically labeled TF proteins, we have demonstrated that all three domains of TF interact with nascent chains during translation. Interactions with the PPIase domain were length-dependent but independent of PPIase activity. Interestingly, with free TF, these same sites were found to be involved in forming the dimer interface, suggesting that dimerization partially occludes TF-nascent chain binding sites. Our data indicate the existence of two regions on TF along which nascent chains can interact, the NC-domains as the main site and the PPIase domain as an auxiliary site.     

Effect of C-terminal truncation on the molecular chaperone function and dimerization of Escherichia coli trigger factor

To examine the role of the C-terminal domain in the chaperone function of trigger factor (TF), a number of truncation mutants were constructed, namely: TF419, TF389, TF380, TF360, TF344, and TF251, in which the C-terminal 13, 43, 52, 72, 88 residues or the entire C-domain were deleted, respectively. Co-expression of mutant chicken adenylate kinase (AK) with TF and the C-terminal truncation mutants was achieved using a plasmid pBVAT that allows expression of TF and AK from a single plasmid. The results show that truncation of the C-terminus of TF has only minor effect on its ability to assist AK refolding in vivo. Further, ribosome-binding experiments indicate that C-terminal truncation mutants can still bind to the ribosome and the presence of the C-terminus may in fact lower the affinity of TF for the ribosome in vivo. This indicates that the C-domain of trigger factor may not be essential for the ribosome-associated molecular chaperone function of TF. However, the purified TF C-terminal truncation mutants had a dramatically reduced ability to assist rabbit muscle GAPDH refolding in vitro and a reduced tendency to dimerize. This shows that the structural integrity of the C-terminus contributes to both the chaperone function of TF and the stability of the dimeric form.     

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.     

Mode of action of the antiprion drugs 6AP and GA on ribosome assisted protein folding

The ribosome, the protein synthesis machinery of the cell, has also been implicated in protein folding. This activity resides within the domain V of the main RNA component of the large subunit of the ribosome. It has been shown that two antiprion drugs 6-aminophenanthridine (6AP) and Guanabenz (GA) bind to the ribosomal RNA and inhibit specifically the protein folding activity of the ribosome. Here, we have characterized with biochemical experiments, the mode of inhibition of these two drugs using ribosomes or ribosomal components active in protein folding (referred to as ’ribosomal folding modulators’ or RFMs) from both bacteria Escherichia coli and yeast Saccharomyces cerevisiae, and human carbonic anhydrase (HCA) as a sample protein. Our results indicate that 6AP and GA inhibit the protein folding activity of the ribosome by competition with the unfolded protein for binding to the ribosome. As a result, the yield of the refolded protein decreases, but the rate of its refolding remains unaffected. Further, 6AP- and GA mediated inhibition of RFM mediated refolding can be reversed by the addition of RFMs in excess. We also demonstrate with delayed addition of the ribosome and the antiprion drugs that there is a short time-span in the range of seconds within which the ribosome interacts with the unfolded protein. Thus we conclude that the protein folding activity of the ribosome is conserved from bacteria to eukaryotes and most likely the substrate for RFMs is an early refolding state of the target protein.     

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.     

Functional adaptations of the bacterial chaperone trigger factor to extreme environmental temperatures

Trigger factor (TF) is the first molecular chaperone interacting cotranslationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. Thermal adaptation of chaperone function was investigated in TFs from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by bacteria. Although structurally homologous, these TFs display strikingly distinct properties that are related to the bacterial environmental temperature. The hyperthermophilic TF strongly binds model proteins during their folding and protects them from heat‐induced misfolding and aggregation. It decreases the folding rate and counteracts the fast folding rate imposed by high temperature. It also functions as a carrier of partially folded proteins for delivery to downstream chaperones ensuring final maturation. By contrast, the psychrophilic TF displays weak chaperone activities, showing that these functions are less important in cold conditions because protein folding, misfolding and aggregation are slowed down at low temperature. It efficiently catalyses prolyl isomerization at low temperature as a result of its increased cellular concentration rather than from an improved activity. Some chaperone properties of the mesophilic TF possibly reflect its function as a cold shock protein in E. coli.     

The various facets of the protein-folding activity of the ribosome

In addition to its involvement in protein synthesis, the ribosome is implicated in protein folding. Some co-translational events, such as the rhythm of protein synthesis, the passage through the exit tunnel of the ribosome, or the interaction with ribosome-associated chaperones may help protein folding. Ribosomes from prokaryotes, eukaryotes, and mitochondria have also been shown to assist the folding of denatured proteins in vitro in a translation-independent way. This intriguing protein-folding activity of the ribosome (PFAR, also termed RPFA) has been mapped to the domain V of the large rRNA of the large subunit of the ribosome. Unfolded, newly synthesized proteins catalyze the dissociation of the two ribosomal subunits in vitro, thereby promoting ribosome recycling and facilitating accessibility of domain V to these proteins, which in turn may help their folding by PFAR. The recent identification of 6AP and GA - the two first drugs that specifically inhibit PFAR without affecting protein translation - will help decipher the biological significance of PFAR in vivo. Of note, 6AP and GA were initially isolated on the basis of their activity against prion-based diseases. Recently, 6AP and GA were also shown to be active in vivo in a drosophila model for oculopharyngeal muscular dystrophy, which is another amyloid-based disease. This effect is mimicked by large deletions in the ribosomal DNA (rDNA) locus. In addition, small deletions in the rDNA locus show a synergistic effect with low doses of 6AP and GA. Hence, PFAR may be involved in various amyloid-based diseases.     

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.     

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.     

Cotranslational Folding of Proteins

We suppose that folding of proteins occurs cotranslationally by the following scheme. The polypeptide chains enter the folding sites from protein translocation complexes (ribosome, translocation machinery incorporated in membranes) directionally with the N-terminus and gradually. The chain starts to fold as soon as its N-terminal residue enters the folding site from the translocation complex. The folding process accompanies the translocation of the chain to its folding site and is completed after the C-terminal residue leaves the translocation complex. Proteins fold in sequential stages, by translocation of their polypeptide into folding compartments. At each stage a particular conformation of the N-terminal part of the chain that has emerged from the translocation complex is formed. The formation of both the particular conformations of the N-terminal chain segment at each folding stage and the final native protein conformation at the last stage occurs in a time that does not exceed the duration of the fastest elongation cycle on the ribosome.     

A switch from α‐helical to β‐strand conformation during co‐translational protein folding

Cellular proteins begin to fold as they emerge from the ribosome. The folding landscape of nascent chains is not only shaped by their amino acid sequence but also by the interactions with the ribosome. Here, we combine biophysical methods with cryo‐EM structure determination to show that folding of a β‐barrel protein begins with formation of a dynamic α‐helix inside the ribosome. As the growing peptide reaches the end of the tunnel, the N‐terminal part of the nascent chain refolds to a β‐hairpin structure that remains dynamic until its release from the ribosome. Contacts with the ribosome and structure of the peptidyl transferase center depend on nascent chain conformation. These results indicate that proteins may start out as α‐helices inside the tunnel and switch into their native folds only as they emerge from the ribosome. Moreover, the correlation of nascent chain conformations with reorientation of key residues of the ribosomal peptidyl‐transferase center suggest that protein folding could modulate ribosome activity.     

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.     

Ribosome-based protein folding systems are structurally divergent but functionally universal across biological kingdoms

In bacteria, Trigger factor (TF) is the first chaperone that interacts with nascent polypeptides as soon as they emerge from the exit tunnel of the ribosome. TF binds to the ribosomal protein L23 located next to the tunnel exit of the large subunit, with which it forms a cradle-like space embracing the polypeptide exit region. It cooperates with the DnaK Hsp70 chaperone system to ensure correct folding of a number of newly translated cytosolic proteins in Escherichia coli. Whereas TF is exclusively found in prokaryotes and chloroplasts, Saccharomyces cerevisiae, a eukaryotic microorganism, has a three-member Hsp70-J protein complex, Ssb-Ssz-Zuo, which could act as a ribosome-associated folding facilitator. In the work reported in this volume of Molecular Microbiology, Rauch et al. (2005, Mol Microbiol, doi:10.1111/j.1365-2958.2005.04690.x) examined the functional similarity of the ribosome-associated chaperones in prokaryotes and eukaryotes. In spite of the fact that TF and the Hsp70-based triad are structurally unrelated, TF can bind to the yeast ribosome via Rpl25 (the L23 counterpart) and can substitute for some, but not all, of the functions assigned to Ssb-Ssz-Zuo in yeast. The functional conservation of the ribosome-associated chaperones without structural similarity is remarkable and suggests that during evolution nature has employed a common design but divergent components to facilitate folding of polypeptides as they emerge from the ribosomal exit, a fundamental process required for the efficient expression of genetic information.     

Simultaneous binding of trigger factor and signal recognition particle to the E. coli ribosome

Signal recognition particle (SRP) and trigger factor (TF) both bind to ribosomal protein L23 at the peptide exit area on the 50S subunit of the E. coli ribosome. In this study, we have developed a spin-down assay and used it to estimate KD values and the corresponding enthalpies for the binding of radio-labelled SRP and TF to naked ribosomes and to ribosomes carrying a tetrapeptidyl-tRNA in the P site. At 20 degrees C, the KD value for TF binding is 2 microM and for SRP it is 170 nM for naked as well as for translating ribosomes. At 4 degrees C, the KD values for TF and SRP binding are 1.1 microM and 90 nM, respectively. Competition binding experiments reveal that SRP and TF bind simultaneously to the ribosome with little affinity interference, showing that the factors have separate binding sites on L23. This makes an alternating binding mode for TF and SRP less plausible.     

Confined dynamics of a ribosome‐bound nascent globin: Cone angle analysis of fluorescence depolarization decays in the presence of two local motions

We still know very little about how proteins achieve their native three‐dimensional structure in vitro and in the cell. Folding studies as proteins emerge from the mega Dalton‐sized ribosome pose special challenges due to the large size and complicated nature of the ribosome‐nascent chain complex. This work introduces a combination of three‐component analysis of fluorescence depolarization decays (including the presence of two local motions) and in‐cone analysis of diffusive local dynamics to investigate the spatial constraints experienced by a protein emerging from the ribosomal tunnel. We focus on E. coli ribosomes and an all‐α‐helical nascent globin in the presence and absence of the cotranslationally active chaperones DnaK and trigger factor. The data provide insights on the dynamic nature and structural plasticity of ribosome‐nascent chain complexes. We find that the sub‐ns motions of the N‐terminal fluorophore, reporting on the globin dynamics in the vicinity of the N terminus, are highly constrained both inside and outside the ribosomal tunnel, resulting in high‐order parameters (>0.85) and small cone semiangles (<30°). The shorter globin chains buried inside the tunnel are less spatially constrained than those of a reference sequence from a natively unfolded protein, suggesting either that the two nascent chain sequences have a different secondary structure and therefore sample different regions of the tunnel or that the tunnel undergoes local structural adjustments to accommodate the globin sequence. Longer globins emerging out of the ribosomal tunnel are also found to have highly spatially constrained slow (ns) motions. There are no observable spectroscopic changes in the absence of bound chaperones.     

Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding‐induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond‐containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild‐type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding‐induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide‐stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra‐cytosolic compartment, like the periplasm.     

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.