The Escherichia coli thioredoxin homolog YbbN/Trxsc is a chaperone and a weak protein oxidoreductase

Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxin 1 and 2. YbbN, however, does not possess the canonical Cys-x-x-Cys active site of thioredoxins, but instead a Ser-x-x-Cys site. In addition to Cys-38, located in the SxxC site, it contains a second cysteine, Cys-63, close to Cys-38 in the 3D model. Cys-38 and Cys-63 undergo an oxidoreduction process, suggesting that YbbN functions with two redox cysteines. Accordingly, YbbN catalyzes the oxidation of reduced RNase and the isomerization of scrambled RNase. Moreover, upon oxidation, its oligomeric state changes from dimers to tetramers and higher oligomers. YbbN also possesses chaperone properties, promoting protein folding after urea denaturation and forming complexes with unfolded proteins. This is the first biochemical characterization of a member of the YbbN class of bacterial thioredoxin-like proteins, and in vivo experiments will allow to determine the importance of its redox and chaperone properties in the cellular physiology.     

The thioredoxin homolog YbbN functions as a chaperone rather than as an oxidoreductase

Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxins, and possesses a 20 kDa C-terminal part of unknown function. We reported previously that YbbN displays both protein oxido-reductase and chaperone properties in vitro. In this study, we show that an ybbN-deficient strain displays an increased sensitivity to thermal stress but not to oxidative stress, a normal redox state of its cellular proteins but a decreased expression of several cytoplasmic proteins, including EF-Tu, DnaK, GroEL, trigger factor and several Krebs cycle enzymes, suggesting that the chaperone properties of YbbN are more important in vivo than its redox properties. YbbN specifically interacts with DnaK and GroEL, as shown by reverse purification. It increases 4-fold the rate of protein renaturation in vitro by the DnaK chaperone machine, suggesting that it cooperates with DnaK for the optimal expression of several cytoplasmic proteins.     

Structural basis of outer membrane protein biogenesis in bacteria

In Escherichia coli, a multicomponent BAM (β-barrel assembly machinery) complex is responsible for recognition and assembly of outer membrane β-barrel proteins. The functionality of BAM in protein biogenesis is mainly orchestrated through the presence of two essential components, BamA and BamD. Here, we present crystal structures of four lipoproteins (BamB-E). Monomeric BamB and BamD proteins display scaffold architectures typically implied in transient protein interactions. BamB is a β-propeller protein comprising eight WD40 repeats. BamD shows an elongated fold on the basis of five tetratricopeptide repeats, three of which form the scaffold for protein recognition. The rod-shaped BamC protein has evolved through the gene duplication of two conserved domains known to mediate protein interactions in structurally related complexes. By contrast, the dimeric BamE is formed through a domain swap and indicates fold similarity to the β-lactamase inhibitor protein family, possibly integrating cell wall stability in BAM function. Structural and biochemical data show evidence for the specific recognition of amphipathic sequences through the tetratricopeptide repeat architecture of BamD. Collectively, our data advance the understanding of the BAM complex and highlight the functional importance of BamD in amphipathic outer membrane β-barrel protein motif recognition and protein delivery.     

DNA replication defects in a mutant deficient in the thioredoxin homolog YbbN

Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, that displays both redox and chaperone properties. Since three out of the six proteins of the YbbN interactome (Butland et al., 2005) are components of DNA polymerase 3 holoenzyme (i.e. the β-clamp DnaN, the θ subunit HolE and the δ′ subunit HolB), we investigated whether the ybbN mutant presents DNA replication defects. We found that this mutant incorporates ³H-thymidine at higher rates than the parental strain and displays overinitiation, hypermutator and filamentation phenotypes with the occurrence of anucleated cells. Moreover, YbbN functions as a bona fide chaperone in the refolding of the urea-unfolded β-clamp. These results suggest that the DNA replication and cell division defects of the ybbN mutant might best be explained by chaperone functions of YbbN in the biogenesis of DNA polymerase 3 holoenzyme.     

Expression, purification and refolding of the phosphatase domain of protein phosphatase 1 (Ppt1) from Saccharomyces cerevisiae

Here we report the recombinant expression of the catalytically active phosphatase domain of the Saccharomyces cerevisiae protein phosphatase 1 (Ppt1) in E. coli. Ppt1 consists of two domains: a 20 kDa TPR (tetratricopeptide repeat) domain, which mediates protein-protein interactions and directs Ppt1 to potential substrate proteins, e.g. the molecular chaperone Hsp90. The second, a 40 kDa phosphatase domain, exhibits catalytic activity and dephosphorylates phosphorylated serine/threonine residues of respective substrate proteins. The Ppt1 phosphatase domain was cloned and expressed in E. coli in unsoluble inclusion bodies. After isolating these, the aggregates were denatured with guanidinium hydrochloride and soluble protein was purified using affinity chromatography. Optimal renaturation conditions led to large amounts of the refolded phosphatase domain in high purity. Interestingly, further enzymatic studies revealed that the domain is not only correctly folded, but also shows higher catalytic activity compared to the full length protein.     

Crystal Structure of P58(IPK) TPR Fragment Reveals the Mechanism for its Molecular Chaperone Activity in UPR

P58(IPK) might function as an endoplasmic reticulum molecular chaperone to maintain protein folding homeostasis during unfolded protein responses. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promote protein folding within the endoplasmic reticulum, we have determined the crystal structure of P58(IPK) TPR fragment to 2.5 Å resolution by the SAD method. The crystal structure of P58(IPK) revealed three domains (I-III) with similar folds and each domain contains three TPR motifs. An ELISA assay indicated that P58(IPK) acts as a molecular chaperone by interacting with misfolded proteins such as luciferase and rhodanese. The P58(IPK) structure reveals a conserved hydrophobic patch located in domain I that might be involved in binding the misfolded polypeptides. Structure-based mutagenesis for the conserved hydrophobic residues located in domain I significantly reduced the molecular chaperone activity of P58(IPK).     

Structural Basis for the Function of the β-Barrel Assembly-Enhancing Protease BepA

The β-barrel assembly machinery (BAM) complex mediates the assembly of β-barrel membrane proteins in the outer membrane. BepA, formerly known as YfgC, interacts with the BAM complex and functions as a protease/chaperone for the enhancement of the assembly and/or degradation of β-barrel membrane proteins. To elucidate the molecular mechanism underlying the dual functions of BepA, its full-length three-dimensional structure is needed. Here, we report the crystal structure of full-length BepA at 2.6-Å resolution. BepA possesses an N-terminal protease domain and a C-terminal tetratricopeptide repeat domain, which interact with each other. Domain cross-linking by structure-guided introduction of disulfide bonds did not affect the activities of BepA in vivo, suggesting that the function of this protein does not involve domain rearrangement. The full-length BepA structure is compatible with the previously proposed docking model of BAM complex and tetratricopeptide repeat domain of BepA.     

The TPR domain of BepA is required for productive interaction with substrate proteins and the β‐barrel assembly machinery complex

BepA (formerly YfgC) is an Escherichia coli periplasmic protein consisting of an N‐terminal protease domain and a C‐terminal tetratricopeptide repeat (TPR) domain. We have previously shown that BepA is a dual functional protein with chaperone‐like and proteolytic activities involved in membrane assembly and proteolytic quality control of LptD, a major component of the outer membrane lipopolysaccharide translocon. Intriguingly, BepA can associate with the BAM complex: the β‐barrel assembly machinery (BAM) driving integration of β‐barrel proteins into the outer membrane. However, the molecular mechanism of BepA function and its association with the BAM complex remains unclear. Here, we determined the crystal structure of the BepA TPR domain, which revealed the presence of two subdomains formed by four TPR motifs. Systematic site‐directed in vivo photo‐cross‐linking was used to map the protein–protein interactions mediated by the BepA TPR domain, showing that this domain interacts both with a substrate and with the BAM complex. Mutational analysis indicated that these interactions are important for the BepA functions. These results suggest that the TPR domain plays critical roles in BepA functions through interactions both with substrates and with the BAM complex. Our findings provide insights into the mechanism of biogenesis and quality control of the outer membrane.     

Identification of CHIP, a Novel Tetratricopeptide Repeat-Containing Protein That Interacts with Heat Shock Proteins and Negatively Regulates Chaperone Functions

The chaperone function of the mammalian 70-kDa heat shock proteins Hsc70 and Hsp70 is modulated by physical interactions with four previously identified chaperone cofactors: Hsp40, BAG-1, the Hsc70-interacting protein Hip, and the Hsc70-Hsp90-organizing protein Hop. Hip and Hop interact with Hsc70 via a tetratricopeptide repeat domain. In a search for additional tetratricopeptide repeat-containing proteins, we have identified a novel 35-kDa cytoplasmic protein, carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP is highly expressed in adult striated muscle in vivo and is expressed broadly in vitro in tissue culture. Hsc70 and Hsp70 were identified as potential interaction partners for this protein in a yeast two-hybrid screen. In vitro binding assays demonstrated direct interactions between CHIP and both Hsc70 and Hsp70, and complexes containing CHIP and Hsc70 were identified in immunoprecipitates of human skeletal muscle cells in vivo. Using glutathione S-transferase fusions, we found that CHIP interacted with the carboxy-terminal residues 540 to 650 of Hsc70, whereas Hsc70 interacted with the amino-terminal residues 1 to 197 (containing the tetratricopeptide domain and an adjacent charged domain) of CHIP. Recombinant CHIP inhibited Hsp40-stimulated ATPase activity of Hsc70 and Hsp70, suggesting that CHIP blocks the forward reaction of the Hsc70-Hsp70 substrate-binding cycle. Consistent with this observation, both luciferase refolding and substrate binding in the presence of Hsp40 and Hsp70 were inhibited by CHIP. Taken together, these results indicate that CHIP decreases net ATPase activity and reduces chaperone efficiency, and they implicate CHIP in the negative regulation of the forward reaction of the Hsc70-Hsp70 substrate-binding cycle.     

Inactivation of the PKR protein kinase and stimulation of mRNA translation by the cellular co-chaperone P58(IPK) does not require J domain function

P58(IPK) was discovered as an inhibitor of the interferon-induced, protein kinase, PKR. Upon virus infection, PKR can, as part of the host defense system, inhibit mRNA translation by phosphorylating the alpha subunit of protein synthesis eukaryotic initiation factor 2 (eIF-2alpha). We previously found that influenza virus recruits the cellular P58(IPK) co-chaperone to inhibit PKR activity and thus facilitate viral protein synthesis. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs in addition to the highly conserved J domain found in all DnaJ chaperone family members. To define the role of molecular chaperones in regulating cell growth in addition to PKR regulation, we performed a detailed analysis of the P58(IPK) J domain. Using growth rescue assays, we found that the P58(IPK) J domain substituted for the J domains of other DnaJ proteins, including DnaJ in Escherichia coli and Ydj1 in Saccharomyces cerevisiae. This is the first time a cellular J domain from a mammalian DnaJ family member was shown to be functional in both prokaryotic DnaJ and eukaryotic Ydj1 constructs. Furthermore, point mutations within the conserved HPD residue cluster of the P58(IPK) J domain disrupted P58(IPK) J function including stimulation of ATPase activity of Hsp70. However, the P58(IPK) HPD mutants still inhibited PKR activity and thus supported cell growth in a yeast rescue assay. Overexpression of the HPD mutants of P58(IPK), similar to their wild-type counterpart, also stimulated mRNA translation in a mammalian cell system. Taken together, our data necessitate a model of P58(IPK) inhibition of PKR kinase activity and stimulation of mRNA translation, which does not require classical J domain function found in the DnaJ molecular chaperone family.     

Thioredoxin fusions increase folding of single chain Fv antibodies in the cytoplasm of Escherichia coli: evidence that chaperone activity is the prime effect of thioredoxin

In this study we investigate the effect of thioredoxin (Trx1) protein fusions in the production, oxidation, and folding of single chain Fv (scFv) antibodies in the cytoplasm of Escherichia coli. We analyze the expression levels, solubility, disulfide-bond formation, and antigen-binding properties of Trx1-scFv fusions in E. coli wild-type cells and isogenic strains carrying mutations in the glutathione oxidoreductase (gor) and/or thioredoxin reductase (trxB) genes. We compare the Trx1-scFv fusions with other reported systems for production of scFv in the cytoplasm of E. coli, including protein fusions to the maltose-binding protein. In addition, we analyze the effect of co-expressing a signal-sequence-less derivative of the periplasmic chaperone and disulfide-bond isomerase DsbC (DeltassDsbC), which has been shown to act as a chaperone for scFvs in the cytoplasm. The results reported here demonstrate that Trx1 fusions produce the highest expression level and induce the correct folding of scFvs even in the absence of DeltassDsbC in the cytoplasm of E. coli trxB gor cells. The disulfide bridges of Trx1-scFv fusions were formed correctly in E. coli trxB gor cells, but not in trxB single mutants. Antigen-binding assays showed that Trx1 has only a minor influence in the affinity of the scFv, indicating that Trx1-scFv fusions can be used without removal of the Trx1 moiety. In addition, we proved that a Trx1"AGPA" variant, having its catalytic cysteine residues mutated to alanine, was fully capable of assisting the folding of the fused scFvs. Taken together, our data indicate that the Trx1 moiety acts largely as an intramolecular protein chaperone, not as a disulfide bond catalyst, inducing the correct folding of scFvs in the cytoplasm of E. coli trxB gor cells.     

The phosphatase Ppt1 is a dedicated regulator of the molecular chaperone Hsp90

Ppt1 is the yeast member of a novel family of protein phosphatases, which is characterized by the presence of a tetratricopeptide repeat (TPR) domain. Ppt1 is known to bind to Hsp90, a molecular chaperone that performs essential functions in the folding and activation of a large number of client proteins. The function of Ppt1 in the Hsp90 chaperone cycle remained unknown. Here, we analyzed the function of Ppt1 in vivo and in vitro. We show that purified Ppt1 specifically dephosphorylates Hsp90. This activity requires Hsp90 to be directly attached to Ppt1 via its TPR domain. Deletion of the ppt1 gene leads to hyperphosphorylation of Hsp90 in vivo and an apparent decrease in the efficiency of the Hsp90 chaperone system. Interestingly, several Hsp90 client proteins were affected in a distinct manner. Our findings indicate that the Hsp90 multichaperone cycle is more complex than was previously thought. Besides its regulation via the Hsp90 ATPase activity and the sequential binding and release of cochaperones, with Ppt1, a specific phosphatase exists, which positively modulates the maturation of Hsp90 client proteins.     

HBP21: A Novel Member of TPR Motif Family, as a Potential Chaperone of Heat Shock Protein 70 in Proliferative Vitreoretinopathy (PVR) and Breast Cancer

A large number of tetratricopeptide repeat (TPR)-containing proteins have been shown to interact with the C-terminal domain of the 70 kDa heat-shock protein (Hsp70), especially those with three consecutive TPR motifs. The TPR motifs in these proteins are necessary and sufficient for mediating the interaction with Hsp70. Here, we investigate HBP21, a novel human protein of unknown function having three tandem TPR motifs predicted by computational sequence analysis. We confirmed the high expression of HBP21 in breast cancer and proliferative vitreoretinopathy (PVR) proliferative membrane and examined whether HBP21 could interact with Hsp70 using a yeast two-hybrid system and glutathione S-transferase pull-down assay. Previous studies have demonstrated the importance of Hsp70 C-terminal residues EEVD and PTIEEVD for interaction with TPR-containing proteins. Here, we tested an assortment of truncation and amino acid substitution mutants of Hsp70 to determine their ability to bind to HBP21 using a yeast two-hybrid system. The newly discovered interaction between HBP21 and Hsp70 along with observations from other studies leads to our hypothesis that HBP21 may be involved in the inhibition of progression and metastasis of tumor cells. Qinghuai Liu and Juanyu Gao have contributed equally to this work.     

Multiple TPR motifs characterize the Fanconi anemia FANCG protein

The genome protection pathway that is defective in patients with Fanconi anemia (FA) is controlled by at least eight genes, including BRCA2. A key step in the pathway involves the monoubiquitylation of FANCD2, which critically depends on a multi-subunit nuclear 'core complex' of at least six FANC proteins (FANCA, -C, -E, -F, -G, and -L). Except for FANCL, which has WD40 repeats and a RING finger domain, no significant domain structure has so far been recognized in any of the core complex proteins. By using a homology search strategy comparing the human FANCG protein sequence with its ortholog sequences in Oryzias latipes (Japanese rice fish) and Danio rerio (zebrafish) we identified at least seven tetratricopeptide repeat motifs (TPRs) covering a major part of this protein. TPRs are degenerate 34-amino acid repeat motifs which function as scaffolds mediating protein-protein interactions, often found in multiprotein complexes. In four out of five TPR motifs tested (TPR1, -2, -5, and -6), targeted missense mutagenesis disrupting the motifs at the critical position 8 of each TPR caused complete or partial loss of FANCG function. Loss of function was evident from failure of the mutant proteins to complement the cellular FA phenotype in FA-G lymphoblasts, which was correlated with loss of binding to FANCA. Although the TPR4 mutant fully complemented the cells, it showed a reduced interaction with FANCA, suggesting that this TPR may also be of functional importance. The recognition of FANCG as a typical TPR protein predicts this protein to play a key role in the assembly and/or stabilization of the nuclear FA protein core complex.     

The crystal structure of NlpI. A prokaryotic tetratricopeptide repeat protein with a globular fold

There are several different families of repeat proteins. In each, a distinct structural motif is repeated in tandem to generate an elongated structure. The nonglobular, extended structures that result are particularly well suited to present a large surface area and to function as interaction domains. Many repeat proteins have been demonstrated experimentally to fold and function as independent domains. In tetratricopeptide (TPR) repeats, the repeat unit is a helix-turn-helix motif. The majority of TPR motifs occur as three to over 12 tandem repeats in different proteins. The majority of TPR structures in the Protein Data Bank are of isolated domains. Here we present the high-resolution structure of NlpI, the first structure of a complete TPR-containing protein. We show that in this instance the TPR motifs do not fold and function as an independent domain, but are fully integrated into the three-dimensional structure of a globular protein. The NlpI structure is also the first TPR structure from a prokaryote. It is of particular interest because it is a membrane-associated protein, and mutations in it alter septation and virulence.     

The chaperone function of cyclophilin 40 maps to a cleft between the prolyl isomerase and tetratricopeptide repeat domains

Cyclophilin 40 (CyP40), an immunophilin cochaperone present in steroid receptor-Hsp90 complexes, contains an N-terminal peptidylprolyl isomerase (PPIase) domain separated from a C-terminal Hsp90-binding tetratricopeptide repeat (TPR) domain by a 30-residue linker. To map CyP40 chaperone function, CyP40 deletion mutants were prepared and analysed for chaperone activity. CyP40 fragments containing the PPIase domain plus linker or the linker region and the adjoining TPR domain retained chaperone activity, whilst individually, the catalytic and TPR domains were devoid of chaperoning ability. CyP40 chaperone function then, is localized within the linker that forms a binding cleft with potential to accommodate non-native substrates.     

Overlapping sites of tetratricopeptide repeat protein binding and chaperone activity in heat shock protein 90

The sequential binding of different tetratricopeptide repeat (TPR) proteins to heat shock protein 90 (hsp90) is essential to its chaperone function in vivo. We have previously shown that three basic residues in the TPR domain of PP5 are required for binding to the acidic C-terminal domain of hsp90. We have now tested which acidic residues in this C-terminal domain are required for binding to three different TPR proteins as follows: PP5, FKBP52, and Hop. Mutation of Glu-729, Glu-730, and Asp-732 at the C terminus of hsp90 interfered with binding of all three TPR proteins. Mutation of Glu-720, Asp-722, Asp-723, and Asp-724 inhibited binding of FKBP52 and PP5 but not of Hop. Mutation of Glu-651 and Asp-653 did not affect binding of FKBP52 or PP5 but inhibited both Hop binding and hsp90 chaperone activity. We also found that a conserved Lys residue required for PP5 binding to hsp90 was critical for the binding of FKBP52 but not for the binding of Hop to hsp90. These results suggest distinct but overlapping binding sites on hsp90 for different TPR proteins and indicate that the binding site for Hop, which is associated with hsp90 in intermediate stages of protein folding, overlaps with a site of chaperone activity.     

A brain-specific isoform of small glutamine-rich tetratricopeptide repeat-containing protein binds to Hsc70 and the cysteine string protein

Small glutamine-rich tetratricopeptide repeat-containing protein (SGT) is a ubiquitously expressed cochaperone of heat shock cognate protein of 70 kDa (Hsc70). SGT binds to the C terminus of Hsc70, a site used by several tetratricopeptide repeat-containing binding partners to recruit Hsc70 into complexes of diverse function. We describe here an isoform of SGT with 60% amino acid sequence identity that we name betaSGT. In contrast to the previously published alphaSGT, betaSGT is almost exclusively expressed in brain. Both isoforms of SGT possess similar binding properties toward Hsc70 and cysteine string protein, a synaptic vesicle-associated J-domain-containing protein. In addition, SGTs oligomerize without preferences among isoforms. The distribution of protein binding motifs on SGTs reveals a modular structure. The N-terminal domains mediate oligomerization. Binding to Hsc70 is impaired by mutations of basic residues within the central tetratricopeptide repeat domain of betaSGT, indicating a two-carboxylate clamp as the binding mode. The tetratricopeptide repeats are also necessary for binding to the cysteine string protein. However, this binding mode is distinct from the two-carboxylate clamp that is involved in Hsc70 binding. The C-terminal regions of SGTs might constitute independent protein interaction domains. We conclude that betaSGT is likely to cooperate with alphaSGT as co-chaperone of Hsc70 in the brain. The modular structure of SGTs allows them to recruit client proteins to Hsc70 and to direct the resulting complex toward downstream proteins that take over the respective client proteins.