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Originally published In Press as doi:10.1074/jbc.M605164200 on August 22, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31963-31971, October 20, 2006
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The C-terminal Domain of Escherichia coli Trigger Factor Represents the Central Module of Its Chaperone Activity*Formula

Frieder Merz, Anja Hoffmann, Anna Rutkowska, Beate Zachmann-Brand, Bernd Bukau, and Elke Deuerling1

From the Zentrum für Molekulare Biologie der Universität Heidelberg Im Neuenheimer Feld 282, Universität Heidelberg, 69120 Heidelberg, Germany

Received for publication, May 30, 2006 , and in revised form, August 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In bacteria, ribosome-bound Trigger Factor assists the folding of newly synthesized proteins. The N-terminal domain (N) of Trigger Factor mediates ribosome binding, whereas the middle domain (P) harbors peptidyl-prolyl isomerase activity. The function of the C-terminal domain (C) has remained enigmatic due to structural instability in isolation. Here, we have characterized a stabilized version of the C domain (CS), designed on the basis of the recently solved atomic structure of Trigger Factor. Strikingly, only the isolated CS domain or domain combinations thereof (NCS, PCS) revealed substantial chaperone activity in vitro and in vivo. Furthermore, to disrupt the C domain without affecting the overall Trigger Factor structure, we generated a mutant ({Delta}53) by deletion of the C-terminal 53 amino acid residues. This truncation caused the complete loss of the chaperone activity of Trigger Factor in vitro and severely impaired its function in vivo. Therefore, we conclude that the chaperone activity of Trigger Factor critically depends on its C-terminal domain as the central structural chaperone module. Intriguingly, a structurally similar module is found in the periplasmic chaperone SurA and in MPN555, a protein of unknown function. We speculate that this conserved module can exist solely or in combination with additional domains to fulfill diverse chaperone functions in the cell.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A complex network of molecular chaperones controls the folding of newly synthesized proteins in the cytosol. In bacteria, the Trigger Factor (TF),2 because of its location on the ribosome is the first chaperone to assist the folding of newly synthesized proteins. This first encounter is succeeded by the cytosolic Hsp70 and Hsp60 chaperone systems consisting of DnaK, DnaJ, GrpE and GroEL, GroES, respectively (1-3). While the GroEL system is essential for cell viability at all conditions, cells tolerate the absence of TF or DnaK at growth temperatures between 20 and 37 °C. However, the simultaneous deletion of the TF-encoding gene tig and the dnaK gene provokes the aggregation of several hundred cytosolic protein species and the loss of cell viability at temperatures above 30 °C (4-6).

TF is a very abundant protein (50 µM in the cell cytosol) (5) and is present in a 2- to 3-fold excess over ribosomes. It associates in a 1:1 stoichiometry with ribosomes (7, 8), whereas uncomplexed TF is in an equilibrium between a monomeric and dimeric state (9). In vivo, ribosome-associated TF assists the folding of newly synthesized proteins by binding to nascent polypeptides when they emerge from the ribosomal exit tunnel (4, 5, 8, 10-12). In vitro, TF chaperone activity can be monitored by its ability to prevent the aggregation and to promote the refolding of chemically denatured GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (13, 14). Moreover, TF efficiently stimulates the refolding of denatured RNase T1 in vitro (15), where a slow trans to cis isomerization of prolyl residues is the rate-limiting step (16). The high catalytic efficiency of TF in RNase T1 refolding results from two combined activities: the binding of TF as a chaperone and its catalytic activity as a peptidyl-prolyl cis/trans isomerase (PPIase) (17).

Escherichia coli TF consists of 432 amino acids and was biochemically characterized by limited proteolysis to be a three-domain protein (18-21). The N-terminal fragment (aa 1-144) contains a stable domain (N, aa 1-118) that is essential and sufficient for the ribosomal attachment of TF (18). The second domain (P, aa 145-247) displays PPIase activity and shows homology to the PPIase family of FK506-binding proteins (15, 20). The largest domain, which shows no sequence homology to any other known protein family, is formed by the C-terminal segment of TF (C, aa 248-432). No precise function had been assigned to this isolated domain so far, in contrast to the N and P domains. Previous in vitro studies showed that only full-length TF, but none of the isolated domains, displayed chaperone activity toward denatured GAPDH and unfolded RNase T1 (18, 21-23).

The recent crystal structures of E. coli TF revealed that this chaperone has an unusual extended shape (11, 24) and its three domains structurally do not align in a linear manner (Fig. 1). The C-terminal domain of TF builds the center of the molecule with two protruding extensions forming the "arms," whereas the N-terminal and the catalytic PPIase domains localize to opposite distal ends of the protein. Importantly, the structure implies that the C-terminal domain of TF is stabilized by a long N-terminal linker region (aa 112-144), providing an explanation for the instability of the isolated C-terminal domain (aa 248-432) lacking this linker region (18, 21). Furthermore, the structural insights suggest that the central C-terminal domain may strongly contribute to TF chaperone activity. However, recent analyses of TF truncation mutants provide contradicting results: whereas deletions in the C-domain affected the refolding of GAPDH in vitro, this domain was suggested to be dispensable for TF chaperone activity in vivo (25).

To unravel the function of the C-terminal domain and its potential contribution to TF chaperone activity, we designed several new constructs. These comprised either the stabilized C domain, including the N-terminal linker (aa 112-144) or domain combinations thereof. In addition, we truncated the full-length TF by deletion of the 53 C-terminal amino acids. The in vitro and in vivo chaperone activities of these TF variants were characterized in detail by analyzing their capacity to (i) prevent the aggregation and promote the refolding of denatured GAPDH, (ii) refold denatured RNase T1, (iii) form dimers in solution, (iv) cross-link to nascent polypeptides, and (v) complement the synthetic lethality of cells lacking TF and DnaK and prevent the aggregation of cytosolic proteins in these cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Growth Conditions—E. coli strains were derivatives of MC4100. Construction of {Delta}tig{Delta}dnaK S1 and {Delta}dnaK strains were described previously (14, 26). Strains were grown in Luria Bertani (LB) medium containing IPTG as indicated and supplemented with ampicillin (100 µg/ml), spectinomycin (20 µg/ml), or kanamycin (40 µg/ml) where appropriate.

Mutant Construction, Expression, and Protein Purification— TF variants were generated by PCR. The oligonucleotides 5'-ggccggatccatgtatccggaagttgaactgcaggg-3' and 5'-ccggggatcccgcctgctggttcatcagc-3' were used to amplify the PCS and CS fragments. The plasmid pDS56-tig-His6 (18) served as template for the PCS fragment, whereas pDS56-NC (23) was used as template for construction of the CS variant. The C-terminal truncation of 53 amino acids was created using the primers 5'-ggccggatccatgcaagtttcagttgaaacc-3' and 5'-ggccggatccttcgtacgcagaagccatctcttcg-3', whereby pDS56-tig-His6 served as template. All PCR products encoded BamH1 sites on the 5' and 3' ends. These were used for insertion into the vector pDS56 to generate the plasmids pDS56-PCS, pDS56-CS, and pDS56-{Delta}53. The DNA sequence integrity of all mutants was confirmed by sequencing. TF constructs were expressed in E. coli MC4100{Delta}tig (4) as recombinant proteins with a C-terminal His6 tag. Cells were grown in LB medium, and expression was induced with 500 µM IPTG (isopropyl-beta-D-thiogalactoside). TF and variants were purified as described (23). The protein concentration was determined via Bradford assay (Bio-Rad), while the purity was assessed by both SDS-PAGE and mass spectrometry.

GAPDH Activity Assay—The prevention of aggregation and the refolding of denatured GAPDH were performed as described previously (13, 14). In all measurements we used guanidine hydrochloride denatured GAPDH from rabbit muscle (G-2267; Sigma) at a final concentration of 2.5 µM. The final concentrations of TF, TF variants, or BSA varied from 1.25 to 20 µM. Data were fitted to a single-exponential equation (GraFit program). Relative activities of refolded GAPDH were calculated by dividing the respective rate constants by that determined for the same amount of non-denatured GAPDH.

RNase T1 Refolding—The PPIase-dependent refolding of denatured RCM-RNase T1 was performed as previously reported (14, 27). Briefly, RNase T1 from Aspergillus oryzae (R-1003; Sigma) was reduced with dithiothreitol and applied to iodoacetic acid treatment for carboxymethylation. Upon dilution into 1.6 M NaCl, RCM-RNase T1 refolding (1 µM final concentration) was followed by measuring the intrinsic tryptophan fluorescence at 320 nm in a spectrofluorometer (LS55; PerkinElmer Life Sciences) in the presence of TF, TF variants, or BSA (0.2-4 µM). Refolding rates were obtained by fitting the data to a single-exponential equation using the program GraFit. Rates of TF variants are expressed relative to wild-type TF.

Glutaraldehyde Cross-linking—TF and TF variants (5 µg each) were incubated at different concentrations (0.156, 0.625, 2.5, 10, and 20 µM for TF, PCS, NCS, {Delta}53; and 0.625, 2.5, 10, 20, and 40 µM for N, P, CS) in 20 mM Hepes, pH 7.5, 100 mM NaCl, and 1 mM EDTA for 25 min at 25 °C. Cross-linking was initiated by addition of 0.1% glutaraldehyde (Sigma), and the reaction was quenched after 10 min at 25 °C with 100 mM Tris-HCl, pH 7.5, for 15 min at 25 °C. Samples were trichloroacetic acid precipitated (5% trichloroacetic acid, 0.02% NaDOC, 1 h, 4 °C); proteins were pelleted by centrifugation (30 min, 16,000 x g, 4 °C) and pellets resuspended in alkaline sample buffer. SDS-PAGE (12% gel) with Coomassie Brilliant Blue staining was subsequently performed.

Cross-linking to Nascent Polypeptides—An E. coli-based in vitro transcription/translation system derived from MC4100{Delta}tig strain was used (4, 6, 28). To arrest nascent isocitrate dehydrogenase (ICDH) chains at the ribosomes (400 nM) in the presence of TF variants (5 µM), truncated mRNA was generated by adding an antisense oligonucleotide (40 ng/µl; 5'-cccccatctcttcacgcagg-3') and RNase H (0.04 units/µl) to the transcription/translation system. After 20 min of synthesis at 37 °C, translation was stopped by adding 2 mM chloramphenicol. Chemical cross-linking was achieved by addition of 2.5 mM disuccinimidyl suberate and incubation at 25 °C for 30 min. The reaction was quenched with 50 mM Tris-HCl, pH 7.5, for 15 min at 25 °C. Ribosome-nascent chain-TF complexes were isolated by sucrose cushion ultra-centrifugation and subsequently co-immunoprecipitated using a TF-specific antiserum. Isolated [35S]methionine-labeled polypeptides and cross-link products were separated on SDS-PAGE and visualized by autoradiography.

In Vivo Complementation Analysis and Preparation of Aggregates—E. coli {Delta}tig{Delta}dnaK S1 strain was transformed with pDS56 plasmids expressing TF or TF variants under the control of an IPTG-regulatable promoter (18, 26). Expression of TF variants in the absence of IPTG was repressed by LacI encoded on the plasmid pZA4. Cells were grown overnight at 30 °C in the absence of IPTG, diluted to concentrations corresponding to 105, 104, 103, 102, or 10 cells/5 µl, spotted on LB plates containing 0, 10, 20, 50, 100, or 250 µM IPTG, and incubated for 30 h at 30, 34, or 37 °C. Preparation of aggregates was performed according to published procedures (6, 23) from cultures that were grown in LB containing 20 µM IPTG at 30 °C and harvested at an A600 = 1. Experiments were reproduced at least three times.


Figure 1
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  		FIGURE 1.
Structure of TF and TF variants. E. coli Trigger Factor (TF) and TF fragments N, P, CS, NCS, and PCS were modeled according to the structure coordinates of Ref. 11 using the program PyMol and are depicted in ribbon representation. In addition, TF is shown in a space-filling illustration at the left. The C-terminal truncated {Delta}53 mutant was modeled according to the TF structure coordinates from Ref. 24. The N-terminal domain is shown in red, the PPIase is colored in green, and the C-terminal domain is displayed in different shades of blue to emphasize the "body" and the two protruding "arms."

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Design of Trigger Factor Variants—To investigate the contribution of the C-terminal domain to the chaperone activity of TF, we designed several variants (Fig. 1) based on recent crystal structures. The structures suggested that a long N-terminal linker region is important for the three-dimensional organization of the C-terminal domain (11, 24). By fusing this putative stabilizing linker region (aa 112-144) N-terminal to the C terminus (aa 248-432) of TF, we generated a stabilized C-terminal domain (CS). As controls, we included in our analyses the isolated N-terminal domain (N, aa 1-118) and the PPIase domain (P, aa 145-247), which were characterized earlier (18, 23).

Combinations of the CS domain with other TF domains were also investigated. We designed a PCS two-domain fragment comprising amino acids 112-432 and in addition made use of the NC fragment. This was previously generated by fusing the N-terminal 144 amino acids, including the stabilizing linker region, to the C terminus (aa 247-432) (23). The NC fragment is designated hereafter NCS to indicate the presence of the linker region (Fig. 1).

We also constructed a C-terminal truncated TF mutant by deleting the 53 C-terminal amino acids ({Delta}53). The truncation site was designed based on the crystal structure of TF from Vibrio cholerae (24); this version lacks part of the second arm, which results in local rearrangements within the C-terminal domain but has no structural effect on the N or P domain (Fig. 1). All purified TF variants were soluble and thus amenable to functional analysis (data not shown).

The CS Domain Is Essential for the in Vitro Chaperone Activity of TF on Denatured GAPDH—To characterize the in vitro chaperone activity of the various TF fragments, we tested their capacity to prevent aggregation of chemically denatured GAPDH (in 3 M GndHCl) (13, 14, 23). Denatured GAPDH rapidly aggregated upon a 50-fold dilution in non-denaturing reaction buffer as indicated by the increase in light scattering signal at 620 nm. However, the presence of stoichiometric amounts of wild-type TF efficiently prevented GAPDH aggregation as no increase of signal was observed (Fig. 2A). When TF variants were added in a 1:1 ratio, only the PCS fragment inhibited the aggregation of denatured GAPDH by ~50% (Fig. 2A). However, increasing the concentrations of the TF variants revealed that PCS fully suppressed aggregation at a 4-fold (data not shown) or 8-fold excess (20 µM, Fig. 2B) over GAPDH. In addition, at an 8-fold excess NCS reduced aggregation by ~60%. Remarkably, even the isolated CS domain displayed chaperone activity by decreasing GAPDH aggregation by ~15% (Fig. 2B). Neither the individual N nor P domain showed any chaperone activity, whereby the isolated N domain aggregated at high concentrations leading to an increase in the light scattering signal (Fig. 2B). Moreover, {Delta}53 revealed no activity in the prevention of GAPDH aggregation at 20 µM (Fig. 2B) or 40 µM (data not shown). Taken together, these data suggest that an intact C-domain is crucial for the ability of TF to prevent aggregation.

We next investigated whether the TF fragments are able to promote the refolding of GAPDH into its active state. Denatured GAPDH was diluted 50-fold in the presence or absence of TF or TF fragments at different concentrations, and the restored GAPDH activity was determined after 4 h (Fig. 2C). The presence of TF (2.5-5 µM) restored GAPDH activity up to ~55%, whereas a BSA control had no effect. Higher TF concentrations inhibited the refolding of GAPDH as previously reported (13, 23). The addition of increasing concentrations of PCS, NCS, and CS led to increasing total yields of refolded GAPDH up to ~55, 40, and 25%, respectively. However, in contrast to wild-type TF no substantial inhibition of GAPDH refolding was observed (Fig. 2C). Complementing the results above, N, P, and {Delta}53 did not demonstrate any detectable refolding activity at all concentrations tested.


Figure 2
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  		FIGURE 2.
In vitro chaperone activity of TF variants. A and B, aggregation of chemically denatured GAPDH (2.5 µM) was monitored as an increase in light-scattering signal at 620 nm. The prevention of GAPDH aggregation was tested in the presence of 2.5 µM (equimolar concentrations) (A) and 20 µM (B) TF or TF variants. C, refolding of denatured GAPDH was monitored by measuring its enzymatic activity 4 h after 50-fold dilution (final concentration 2.5 µM) in the absence or the presence of different concentrations (1.25, 2.5, 5, 10, and 20 µM) of TF or TF fragments. TF, filled circles; N, open triangles up; P, open triangles down; CS, open circles; NCS, filled triangles up; PCS, filled triangles down; {Delta}53, open diamonds; BSA control, crosses.

 
In summary, all fragments containing the CS domain (NCS, PCS, and CS) showed significant residual chaperone activity although they differed from wild-type TF (i) with a reduced efficiency, and (ii) in their broad effective concentration range for GAPDH refolding. This suggests differences in the interaction of TF, NCS, PCS, and CS with unfolded protein substrates. Furthermore, {Delta}53 lost the ability to prevent the aggregation and to promote the refolding of denatured GAPDH in vitro.We conclude that the CS domain is essential but not sufficient for the full chaperone activity of TF toward denatured GAPDH in vitro.

The CS Domain Is Crucial for Efficient RNase T1 Refolding by TF—A second characteristic in vitro activity of TF is its ability to catalyze the refolding of RNase T1. This process is rate-limited by the slow trans to cis isomerization of peptidyl-prolyl bonds at Pro-39 and Pro-55 (16) and can be followed by measuring the intrinsic tryptophan fluorescence of RNase T1. Wild-type TF in substoichiometric amounts (0.2 µM) efficiently catalyzed the refolding of RNase T1 (1 µM) due to the prolyl isomerase activity of the P domain combined with the capacity to bind substrate as a chaperone (Fig. 3A and Refs. 14, 17, 21). Remarkably, PCS displayed an extremely high catalytic activity with refolding rates similar to full-length TF. In contrast, none of the other TF variants exhibited an efficient catalytic activity even when concentrations of 4 µM were tested (Fig. 3, A and B). The {Delta}53 mutant promoted the refolding of RNase T1 similar to the isolated P domain, suggesting that the requisite complementary chaperone function lies in the C-terminal domain (Fig. 3B). Taken together, these results suggest that the CS domain is essential and sufficient for the binding of the unfolded RNase T1, subsequently enabling the efficient isomerization of the peptidyl-prolyl bonds via the P domain.

Dimer Formation Is Not a Prerequisite for the in Vitro Chaperone Function of TF Variants—Although ribosome-bound TF exerts its function in a monomeric state, previous studies suggest that the in vitro chaperone function of TF in the absence of ribosomes correlates with dimerization (25, 29, 30). To determine whether the chaperone activity of TF variants is dependent on dimer formation, we examined the quaternary structures of the purified TF variants by glutaraldehyde cross-linking (Fig. 4). As previously demonstrated, the appearance of glutaraldehyde cross-linking products reflects the dimerization of TF (9). In agreement with this, wild-type TF showed concentration-dependent dimer formation, with an aberrant SDS-PAGE migration of ~150 kDa. In addition, cross-linking products >170 kDa were observed and most likely represent higher oligomeric or aggregated species of TF generated by glutaraldehyde treatment (9). Although isolated N, P, and CS domains as well as PCS and {Delta}53 revealed no or little dimer formation, the two-domain NCS fragment demonstrated considerable dimerization in a concentration-dependent manner (Fig. 4). This indicates that the N and CS domains together contribute significantly to TF dimer assembly.


Figure 3
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  		FIGURE 3.
RNase T1 refolding by TF variants. Refolding of denatured RNase T1 (1 µM final concentration) was monitored by an increase of intrinsic tryptophan fluorescence at 320 nm after excitation at 268 nm. A, RNase T1 refolding in the absence or presence of 0.2 µM TF or TF variants. B, relative refolding rates of denatured RNase T1 obtained by titration of TF and TF fragments (0, 0.2, 0.4, 1, and 4 µM). TF, filled circles; N, open triangles up; P, open triangles down; CS, open circles; NCS, filled triangles up; PC, filled triangles down; {Delta}53, open diamonds; BSA control, crosses.

 
Importantly, the ability of TF fragments to form dimers did not correlate to their chaperone activity. For example, the PCS fragment was severely impaired in dimer formation and yet showed near wild-type-like activities in GAPDH and RNase T1 refolding (Figs. 2 and 3). Hence, dimer formation is not a pre-requisite for in vitro chaperone activity of the TF variants, suggesting that the monomeric conformation represents an active state.

The CS Domain Shows No Efficient Cross-linking to Nascent Polypeptide Chains—We next sought to investigate the ability of the TF fragments to interact with nascent polypeptide chains. We generated arrested [35S]Met-labeled nascent chains derived from the natural TF substrate ICDH (Fig. 5) in an in vitro transcription/translation system (6) prepared from E. coli cells lacking TF. This enabled the controlled addition of TF variants prior to translation (at 5 µM final concentration to saturate ribosomes) and the subsequent cross-linking of TF to nascent polypeptides via the bifunctional chemical cross-linker disuccinimidyl suberate. In the presence of wild-type TF, two major cross-linking products of ~70 and 90 kDa were obtained (Fig. 5, lane 2) as previously reported (22). In addition, all fragments containing the N-terminal ribosome-binding domain (N, NCS, {Delta}53) efficiently cross-linked to nascent ICDH polypeptides (Fig. 5, lanes 5, 14, 20). As anticipated, no cross-linking to nascent polypeptides was observed for the P domain (6, 23), whereas the PCS and CS fragments showed faint cross-linking products; these are likely weak interactions between these non-ribosomal TF fragments and nascent ICDH mediated by the CS domain. This assumption is supported by the presence of similar faint cross-linking products for wild-type TF when nascent ICDH chains were released from ribosomes by puromycin treatment prior to cross-linker addition (data not shown). Taken together, although PCS and CS are active as chaperones in vitro, an efficient interaction with nascent polypeptides requires the ribosome-binding domain (N), a finding that is in agreement with previous results (18, 23).


Figure 4
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  		FIGURE 4.
Dimerization of TF variants. Increasing concentrations (indicated by triangular gradient symbols) of purified TF or TF variants (0.156, 0.625, 2.5, 10, and 20 µM for TF, PCS,NCS, {Delta}53 and 0.625, 2.5, 10, 20, and 40 µM for N, P, CS) were cross-linked with 0.1% glutaraldehyde, trichloroacetic acid, precipitated, and separated by 12% SDS-PAGE. Equal amounts of protein were loaded on each lane (5 µg). The first lane of each TF variant was not treated with glutaraldehyde. Cross-linked dimers are marked by stars. Higher molecular mass cross-links represent higher oligomeric or aggregated species (see "Results").

 


Figure 5
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  		FIGURE 5.
Interaction of TF and TF fragments with nascent polypeptides. Ribosome-arrested 35S-labeled nascent polypeptides of ICDH (aa 1-177) were generated in an in vitro transcription/translation system supplemented with TF or TF fragments. After synthesis, an aliquot was taken (S) and the residual was subjected to chemical cross-linking by the addition of disuccinimidyl suberate. After disuccinimidyl suberate treatment ribosome-nascent chain complexes were purified by sucrose cushion centrifugation (X) and subsequently co-immunoprecipitated where indicated (IP) to identify cross-link products of TF and TF variants to ICDH (177 aa, indicated by stars). Additional weak cross-linked bands that appear in the background are of unknown identity.

 


Figure 6
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  		FIGURE 6.
Analyses of growth and protein aggregation of {Delta}tig{Delta}dnaK S1 cells expressing TF variants. A, growth analysis of wild-type MC4100, {Delta}dnaK, and {Delta}tig{Delta}dnaK S1 cells expressing TF and TF fragments at different temperatures in the presence of varying amounts of IPTG. Cells were grown overnight at 30 °C and after dilution (corresponding to 105, 104, 103, 102, or 10 cells/spot) spotted on LB plates (containing 0-250 µM IPTG) and incubated 30 h at the indicated temperatures. B, for aggregation analysis cells were grown at 30 °C in LB medium in the presence of 20 µM IPTG. At log-phase (A600 = 1), cells were harvested and aggregates were isolated and analyzed by SDS-PAGE and Coomassie Brilliant Blue staining.

 
The CS Domain Is Important for TF Chaperone Function in Vivo—To examine the in vivo functionality of TF variants, we used an E. coli MC4100 {Delta}tig{Delta}dnaK S1 strain lacking TF and DnaK. These cells are viable at temperatures up to 30 °C due to an unknown suppressor mutation (S1) but cannot grow at higher temperatures. This characteristic provided a means to test the in vivo functionality of the TF variants (26). The {Delta}tig{Delta}dnaK S1 strain was transformed with plasmids expressing TF variants under the control of an IPTG-inducible promoter. In liquid culture the expression levels of each TF variant were similar at each particular IPTG concentration tested (see supplemental Fig. S1). Freshly generated transformants and control cells (MC4100 wild-type and MC4100 {Delta}dnaK) were grown overnight without IPTG at 30 °C, spotted in serial dilutions on LB plates containing different concentrations of IPTG, and incubated for 30 h at 30, 34, and 37 °C (Fig. 6A). {Delta}tig{Delta}dnaK S1 cells containing the vector plasmid grew exclusively at 30 °C, whereas cells expressing wild-type TF grew in an IPTG-dependent manner up to 37 °C, reflecting the growth limit of cells lacking DnaK (Fig. 6A) (4, 31). The TF-complemented {Delta}tig{Delta}dnaK S1 strain grew at 34 °C without the addition of inducer due to the leakiness of the IPTG-controlled promoter. High overexpression of TF resulted in loss of cell viability for unknown reasons as reported earlier (23). Under stringent conditions at 37 °C (Fig. 6A, lower panel), only NCS and PCS in addition to wild-type TF were able to complement the growth deficiency of {Delta}tig{Delta}dnaK S1 cells. Remarkably, NCS complemented the growth in a similar manner to wild-type TF, whereas the PCS fragment lacking the ribosome-binding domain was clearly less efficient. At lower temperature (Fig. 6A, 34 °C, middle panel), high overexpression of the N domain or the CS domain (100 and 250 µM IPTG) also slightly enhanced cell viability. Moreover, the {Delta}53 variant supported growth of {Delta}tig{Delta}dnaK S1 cells at 34 °C, albeit less efficiently than wild-type TF as judged by the number of colony-forming units. Importantly, the observed complementation of the TF variants expressed in {Delta}tig{Delta}dnaK S1 cells resulted from a residual in vivo TF activity rather than from an indirect effect of the unknown suppressor mutation (S1); all fragments that supported the growth of {Delta}tig{Delta}dnaK S1 cells also enabled the simultaneous deletion of the tig and dnaK genes at 30 °C in MC4100 wild-type cells (data not shown).


Figure 7
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  		FIGURE 7.
The C terminus of TF is a putative chaperone module. TF, SurA, and MPN555 share the same structural features (11, 35, 36) in their putative chaperone active domain, composed of a single helix (green), a two-helix arm (red), and a three-helix arm (blue).

 
Furthermore, we investigated the in vivo chaperone function of TF variants by comparing their capacity to prevent the aggregation of cytosolic proteins in {Delta}tig{Delta}dnaK S1 cells (Fig. 6B). The analysis was performed under growth conditions in which all cells expressing different TF variants were viable and comparable in growth (30 °C, 20 µM IPTG). Minor protein aggregation was detected in cells lacking DnaK compared with wild-type cells (6, 26), whereas pronounced aggregation of cytosolic proteins was found in {Delta}tig{Delta}dnaK S1 cells carrying the vector plasmid (Fig. 6B, lanes 1-3). In accordance with earlier findings, the majority of the aggregation-prone cytosolic proteins had a molecular mass larger than 40 kDa (6, 26). Expression of wild-type TF in {Delta}tig{Delta}dnaK S1 cells efficiently reduced protein aggregation to a level comparable with {Delta}dnaK cells. Moreover, the NCS, PCS, and CS fragments decreased protein aggregation in {Delta}tig{Delta}dnaK S1 cells, albeit to a different extent and less efficiently than wild-type TF. In contrast, the expression of N, P, or {Delta}53 was ineffective or even enhanced the protein aggregation. These findings suggest that TF fragments lacking an intact CS domain lose their ability to prevent protein aggregation in vivo.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The function of the C-domain of TF has remained enigmatic for many years as it was unstable when expressed in isolation (18, 21). We have shown here that the addition of the N-terminal linker region stabilizes the C-terminal domain (CS) and alleviates this problem. In addition, we truncated the C terminus by 53 amino acids ({Delta}53) to investigate the consequences on TF functionality. We thoroughly tested CS, domain combinations thereof, and {Delta}53 for both their in vitro chaperone activity and their in vivo functionality. These analyses revealed that the C-terminal domain constitutes the central chaperone module of TF.

In vitro, we found that the CS domain possesses a chaperone activity on its own. Although this activity was lower than that of wild-type TF, the CS domain displayed a similar functionality to prevent aggregation and even promote the refolding of denatured GAPDH into its active state (Fig. 2). Importantly, while none of the other individual domains of TF (N, P) exhibited any chaperone activity, the two-domain fragments (NCS and PCS) showed a conspicuously enhanced chaperone activity as compared with the CS domain alone. Thus, although the N and P domains contribute, the CS is essential for the chaperone activity of TF. This conclusion is supported by the finding that truncation of the C terminus ({Delta}53) resulted in the complete loss of TF in vitro chaperone activity, evident in the inability of {Delta}53 to prevent aggregation or promote GAPDH refolding.

The predominant role of the CS domain in TF chaperone activity is also apparent in the in vitro RNase T1 refolding analyses. Efficient refolding of denatured RNase T1 requires both the PPIase activity of the P domain and the chaperone activity for tight substrate binding (17). The two-domain fragment PCS revealed a wild-type-like TF activity in RNase T1 refolding, whereas only a low level of refolding was observed for the isolated P domain. Thus, the major chaperone activity required for efficient RNase T1 refolding can be assigned to the CS domain. This is further supported by the observation that the {Delta}53 mutant and the isolated P domain have similarly low refolding activities. Deletion of the C-terminal 53 amino acid residues is expected to disturb the structural arrangement of the C-domain alone (Fig. 1) while preserving the fold of the N and P domains. The V. cholerae structure (24) of a similar truncated TF reveals that the partial deletion of the second "arm" causes a collapse of the C-terminal domain, such that the first "arm" has a closer proximity to the N-terminal domain and a loop of the C-domain inserts into the P domain. This insertion in the P domain does not affect its activity, as {Delta}53 demonstrated a PPIase activity in RNase T1 refolding comparable with the isolated P domain (Fig. 3). Thus, it is unlikely that the disturbance of the C-domain causes severe functional defects in the P or N domain, and the observed chaperone deficiency of {Delta}53 is predominantly due to the loss of the C-terminal domain activity.

In vivo, all TF variants containing the N-domain necessary for ribosome association (N, NCS, and {Delta}53) compensated to some degree for the loss of TF in {Delta}tig{Delta}dnaK S1 cells, in line with previous findings (23, 32). Surprisingly, we found that PCS and, to a minor extent, CS were also able to support growth of cells lacking TF and DnaK at 34 °C (Fig. 6A). These fragments cannot associate with ribosomes (data not shown) and did not interact efficiently with nascent chains as shown by the cross-linking experiments (Fig. 5). How can these results be explained in light of earlier data (10) showing that the in vivo activity of full-length TF critically depends on ribosome association? Perhaps the weak interaction of the PCS and CS fragments with nascent polypeptides (Fig. 5) might be sufficient to promote the growth of {Delta}tig{Delta}dnaK cells. Another plausible explanation is that these fragments reveal a cytosolic chaperone activity distinct from the activity of wild-type TF. Such a modified chaperone activity might be even more apparent due to the monomeric state of these fragments and/or the absence of domains that could interfere with accessibility of substrate binding sites in the C domain. This hypothesis is supported by the observation that, whereas PCS and wild-type TF display similar in vitro chaperone functionality, PCS did not demonstrate wild-type-like inhibition on GAPDH refolding at high concentrations. This suggests differences between PCS and TF in substrate interaction (Fig. 2C). Finally, another explanation is that TF per se has a cytosolic chaperone activity that may be more evident in the PCS and CS fragments due to their inability to associate with ribosomes. However, in contrast to PCS and CS, only wild-type TF and the NCS fragment fully complemented the growth defects of {Delta}tig{Delta}dnaK S1 cells up to 37 °C, which underscores the importance of the ribosome association of TF for its in vivo function.

Notably, the ability of TF fragments to complement the growth of {Delta}tig{Delta}dnaK S1 cells did not necessarily correlate with their capacity to prevent the aggregation of cytosolic proteins (Fig. 6, A and B). In particular, the {Delta}53 mutant showed complementation up to 34 °C but failed to prevent protein aggregation in vivo. This is consistent with the in vitro analyses showing that the destruction of the C terminus in {Delta}53 causes the loss of chaperone activity. How can the {Delta}53 variant promote the growth of {Delta}tig{Delta}dnaK S1 cells without preventing protein aggregation? Recent in vitro studies showed that TF in complex with the ribosome functions as a protective shield to prevent untimely degradation of unfolded nascent polypeptides by proteases (33, 34). It is tempting to speculate that the {Delta}53 mutant, which has no detectable chaperone activity but still efficiently associated with ribosomes and nascent polypeptides (Fig. 5), may still provide protection at the ribosome and thereby complement the loss of TF to some degree. To test this hypothesis, we analyzed the capacity of {Delta}53 to protect nascent ICDH polypeptide chains against degradation by proteinase K in vitro (supplemental Fig. S2). Indeed, the proteolytic digestion of nascent ICDH polypeptides was retarded in the presence of {Delta}53. However, we also observed degradation of {Delta}53 itself during proteinase K treatment. Thus, we are not able to unambiguously assign a shielding function for {Delta}53, as part of the protective effect might be due to a substrate competition for proteinase K.

Interestingly, NCS revealed a shielding activity similar to wild-type TF, whereas PCS did not (33) (supplemental Fig. S2). Although the in vitro chaperone activity of NCS is less than that of PCS, this protective ability provides an explanation for how NCS can both fully support {Delta}tig{Delta}dnaK S1 cell growth and reduce in vivo protein aggregation. In general, all TF fragments containing the CS domain reduced the amount of protein aggregation in {Delta}tig{Delta}dnaK S1 cells (NCS, PCS, and to a lesser extent CS). Taken together, these results indicate that the CS domain is the central module of TF chaperone activity in vitro and in vivo.

Interestingly, two other proteins, E. coli SurA (35) and M. pneumoniae MPN555 (36), possess domains with structural homology to the C-terminal domain of TF (11, 36) despite no relevant similarity at the amino acid level (Fig. 7). MPN555 protein is a single-domain protein with unknown function. SurA is a periplasmic chaperone dedicated to the folding of outer membrane porins in Gram-negative bacteria with two PPIase domains in addition to the TF CS-like domain. A SurA fragment lacking these two PPIase domains was found to be sufficient for SurA chaperone function in vitro and in vivo (37). The structural similarities of the C-terminal domain of TF to SurA and MPN555, together with the finding that this domain exists as isolated protein (MPN555) or can act in isolation (CS of TF and SurA mutant lacking the PPIase domains), suggest that it represents a chaperone module on its own. The activity of this chaperone module can be modified by the addition of other domains to fulfill the appropriate function in the periplasm, as evident for SurA, or at the ribosome, in the case of TF.


   FOOTNOTES
 
* This work was supported by DFG Grant SFB638 (to B. B. and E. D.), by the Human Frontiers in Science Program (to E. D.), a Heisenberg Fellowship of the DFG (to E. D.), a Fonds der Chemischen Industrie grant (to B. B.), and by a Boehringer Ingelheim Fonds scholarship (to A. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. Back

1 To whom correspondence should be addressed. Tel.: 49-6221-546870; Fax: 49-6221-545894; E-mail: e.deuerling{at}zmbh.uni-heidelberg.de.

2 The abbreviations used are: TF, Trigger Factor; PPIase, peptidyl-prolyl cis/trans isomerase; N, N-terminal domain of Trigger Factor; P, PPIase domain of Trigger Factor; C, C-terminal domain of Trigger Factor; Cs, stabilized C-terminal domain of Trigger Factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICDH, isocitrate dehydrogenase; IPTG, isopropyl 1-thio-beta-D-galactopyranoside; LB, Luria Bertani; aa, amino acid; BSA, bovine serum albumin. Back


   ACKNOWLEDGMENTS
 
We thank members of the Deuerling and Bukau laboratories, in particular Renee D. Wegrzyn and Heather Sadlish, for comments on the manuscript and discussions. We thank Nenad Ban, Timm Maier, and Lars Ferbitz for their valuable expertise, Christian Graf and Fernanda Rodriguez for analyses by mass spectrometry, and Steffen Preissler and Sebastian Specht for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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