Dedicated metallochaperone connects apoenzyme and molybdenum cofactor biosynthesis components

The biogenesis of molybdenum-containing enzymes is a sophisticated process involving the insertion of a complex molybdenum cofactor into competent apoproteins. As for many molybdoenzymes, the maturation of trimethylamine-oxide reductase TorA requires a private chaperone. This chaperone (TorD) interacts with the signal peptide and the core of apo-TorA. Using random mutagenesis, we established that alpha-helix 5 of TorD plays a key role in the core binding and that this binding drives the maturation of TorA. In addition, we showed for the first time that TorD interacts with molybdenum cofactor biosynthesis components, including MobA, the last enzyme of cofactor synthesis, and Mo-molybdopterin, the precursor form of the cofactor. Finally we demonstrated that TorD also binds the mature molybdopterin-guanine dinucleotide form of the cofactor. We thus propose that TorD acts as a platform connecting the last step of the synthesis of the molybdenum cofactor just before its insertion into the catalytic site of TorA.     

Coexpression of TorD enhances the transport of GFP via the TAT pathway

Twin-arginine translocation (Tat) pathway is capable of secreting fully folded proteins into the periplasm of Gram-negative bacteria and may thus be an ideal system for the expression of active cofactor-containing proteins. However, the applications of Tat system for such purpose have been plagued by low translocation efficiencies. In this study, we demonstrate that the coexpression of a soluble chaperone, TorD, in conjunction with the TorA signal peptide, the translocation efficiency of GFP can be enhanced by more than three-fold. The enhancement in translocation efficiency is believed to be a result of reduced proteolysis mediated by the binding of TorD toward the TorA signal peptide. We believe this approach can be further exploited for the expression and secretion of other heterologous proteins as well as traditional Tat substrate proteins.     

TorD, an essential chaperone for TorA molybdoenzyme maturation at high temperature

TorD has been recognized as an accessory protein that improves maturation of TorA, the molybdenum cofactor-containing trimethylamine oxide reductase of Escherichia coli. In this study, we show that at 42 degrees C and in the absence of TorD TorA is poorly matured and almost completely degraded. Strikingly, TorD restores TorA maturation to the same level whatever the growth temperature. In vitro experiments in which apoTorA was incubated with or without TorD at various temperatures confirm that TorD is an essential chaperone for TorA at elevated temperatures preventing apoTorA mis-folding before cofactor insertion.     

Coordinating assembly and export of complex bacterial proteins

The Escherichia coli twin-arginine protein transport (Tat) system is a molecular machine dedicated to the translocation of fully folded substrate proteins across the energy-transducing inner membrane. Complex cofactor-containing Tat substrates, such as the model (NiFe) hydrogenase-2 and trimethylamine N-oxide reductase (TorA) systems, acquire their redox cofactors prior to export from the cell and require to be correctly assembled before transport can proceed. It is likely, therefore, that cellular mechanisms exist to prevent premature export of immature substrates. Using a combination of genetic and biochemical approaches including gene knockouts, signal peptide swapping, complementation, and site-directed mutagenesis, we highlight here this crucial 'proofreading' or 'quality control' activity in operation during assembly of complex endogenous Tat substrates. Our experiments successfully uncouple the Tat transport and cofactor-insertion activities of the TorA-specific chaperone TorD and demonstrate unequivocally that TorD recognises the TorA twin-arginine signal peptide. It is proposed that some Tat signal peptides operate in tandem with cognate binding chaperones to orchestrate the assembly and transport of complex enzymes.     

Features of a twin-arginine signal peptide required for recognition by a Tat proofreading chaperone

The twin-arginine translocation (Tat) system is a bacterial protein targeting pathway. Tat-targeted proteins display signal peptides containing a distinctive SRRxFLK ‘twin-arginine’ motif. The Escherichia coli trimethylamine N-oxide reductase (TorA) bears a bifunctional Tat signal peptide, which directs protein export and serves as a binding site for the TorD biosynthetic chaperone. Here, the physical interaction between TorD and the TorA signal peptide was investigated. A single substitution within the TorA signal peptide (L31Q) was sufficient to impair TorD binding. Screening of a random torD mutant library identified a variant TorD protein (Q7L) that displayed increased binding affinity for the TorA signal peptide.

Structured summary

MINT-6796225, MINT-6796279, MINT-6796298, MINT-6796315, MINT-6796332, MINT-6796350, MINT-6796371, MINT-6796391, MINT-6796410, MINT-6796429, MINT-6796446, MINT-6796460:

TorD (uniprotkb:P36662) physically interacts (MI:0218) with TorA (uniprotkb:P33225) by two-hybrid (MI:0018)

MINT-6796515, MINT-6796563, MINT-6796589, MINT-6796624, MINT-6796648, MINT-6796666, MINT-6796770, MINT-6796750:

TorA (uniprotkb:P33225) binds (MI:0407) to TorD (uniprotkb:P36662) by isothermal titration calorimetry (MI:0065)

     

Quality control of a molybdoenzyme by the Lon protease

Molybdoenzymes contain a molybdenum cofactor in their active site to catalyze various redox reactions in all domains of life. To decipher crucial steps during their biogenesis, the TorA molybdoenzyme of Escherichia coli had played a major role to understand molybdoenzyme maturation process driven by specific chaperones. TorD, the specific chaperone of TorA, is also involved in TorA protection. Here, we show that immature TorA (apoTorA) is degraded in vivo and in vitro by the Lon protease. Lon interacts with apoTorA but not with holoTorA. Lon and TorD compete for apoTorA binding but TorD binding protects apoTorA against degradation. Lon is the first protease shown to eliminate an immature or misfolded molybdoenzyme probably by targeting its inactive catalytic site.     

Involvement of a mate chaperone (TorD) in the maturation pathway of molybdoenzyme TorA

As many prokaryotic molybdoenzymes, the trimethylamine oxide reductase (TorA) of Escherichia coli requires the insertion of a bis(molybdopterin guanine dinucleotide)molybdenum cofactor in its catalytic site to be active and translocated to the periplasm. We show in vitro that the purified apo form of TorA was activated weakly when an appropriate bis(molybdopterin guanine dinucleotide)molybdenum source was provided, whereas addition of the TorD chaperone increased apoTorA activation up to 4-fold, allowing maturation of most of the apoprotein. We demonstrate that TorD alone is sufficient for the efficient activation of apoTorA by performing a minimal in vitro assay containing only the components for the cofactor synthesis, apoTorA and TorD. Interestingly, incubation of apoTorA with TorD before cofactor addition led to a significant increase of apoTorA activation, suggesting that TorD acts on apoTorA before cofactor insertion. This result is consistent with the fact that TorD binds to apoTorA and probably modifies its conformation in the absence of cofactor. Therefore, we propose that TorD is involved in the first step of TorA maturation to make it competent to receive the cofactor.     

Chaperone protection of immature molybdoenzyme during molybdenum cofactor limitation

Maturation of molybdoenzyme TorA involves chaperone TorD. This study shows that TorD is also required to protect apoTorA against proteolysis when the molybdenum cofactor is limiting in Escherichia coli. The absence of TorD leads to a complete loss of apoTorA during molybdenum cofactor deficiency whereas the presence of TorD maintains a significant amount of apoTorA that can be matured when the molybdenum cofactor becomes available.     

The hydrophobic core of twin-arginine signal sequences orchestrates specific binding to Tat-pathway related chaperones

Redox enzyme maturation proteins (REMPs) bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD (specific for dimethylsulfoxide reductase, DmsA) and TorD (specific for trimethylamine N-oxide reductase, TorA). Green fluorescent protein (GFP) was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre-proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function.     

A genetic screen for suppressors of Escherichia coli Tat signal peptide mutations establishes a critical role for the second arginine within the twin-arginine motif

The Escherichia coli Tat protein export pathway transports folded proteins synthesized with N-terminal twin-arginine signal peptides. Twin-arginine signal sequences contain a conserved SRRxFLK "twin-arginine" amino acid sequence motif which is required for protein export by the Tat pathway. The E. coli trimethylamine N-oxide reductase (TorA) is a Tat-dependent periplasmic molybdoenzyme that facilitates anaerobic respiration with trimethylamine N-oxide as terminal electron acceptor. Here, we describe mutant strains constructed with modified TorA twin-arginine signal peptides. Substitution of the second arginine residue of the TorA signal peptide twin-arginine motif with either lysine or aspartate, or the simultaneous substitution of both arginines with lysine residues, completely abolished export. In each case, the now cytoplasmically localised TorA retained full enzymatic activity with the artificial electron donor benzyl viologen. However, the mutant strains were incapable of anaerobic growth with trimethylamine N-oxide and the non-fermentable carbon-source glycerol. The growth phenotype of the mutant strains was exploited in a genetic screen with the aim of identifying second-site suppressor mutations that allowed export of the modified TorA precursors.     

Competition between Sec- and TAT-dependent protein translocation in Escherichia coli.

Recently, a new protein translocation pathway, the twin-arginine translocation (TAT) pathway, has been identified in both bacteria and chloroplasts. To study the possible competition between the TAT- and the well-characterized Sec translocon-dependent pathways in Escherichia coli, we have fused the TorA TAT-targeting signal peptide to the Sec-dependent inner membrane protein leader peptidase (Lep). We find that the soluble, periplasmic P2 domain from Lep is re-routed by the TorA signal peptide into the TAT pathway. In contrast, the full-length TorA-Lep fusion protein is not re-routed into the TAT pathway, suggesting that Sec-targeting signals in Lep can override TAT-targeting information in the TorA signal peptide. We also show that the TorA signal peptide can be converted into a Sec-targeting signal peptide by increasing the hydrophobicity of its h-region. Thus, beyond the twin-arginine motif, the overall hydrophobicity of the signal peptide plays an important role in TAT versus Sec targeting. This is consistent with statistical data showing that TAT-targeting signal peptides in general have less hydrophobic h-regions than Sec-targeting signal peptides.     

Genetic analysis of pathway specificity during posttranslational protein translocation across the Escherichia coli plasma membrane

In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the "Sec avoidance signal," the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.     

Conserved signal peptide recognition systems across the prokaryotic domains

The twin-arginine translocation (Tat) pathway is a protein targeting system found in bacteria, archaea, and chloroplasts. Proteins are directed to the Tat translocase by N-terminal signal peptides containing SRRxFLK "twin-arginine" amino acid motifs. The key feature of the Tat system is its ability to transport fully folded proteins across ionically sealed membranes. For this reason the Tat pathway has evolved for the assembly of extracytoplasmic redox enzymes that must bind cofactors, and so fold, prior to export. It is important that only cofactor-loaded, folded precursors are presented for export, and cellular processes have been unearthed that regulate signal peptide activity. One mechanism, termed "Tat proofreading", involves specific signal peptide binding proteins or chaperones. The archetypal Tat proofreading chaperones belong to the TorD family, which are dedicated to the assembly of molybdenum-dependent redox enzymes in bacteria. Here, a gene cluster was identified in the archaeon Archaeoglobus fulgidus that is predicted to encode a putative molybdenum-dependent tetrathionate reductase. The gene cluster also encodes a TorD family chaperone (AF0160 or TtrD) and in this work TtrD is shown to bind specifically to the Tat signal peptide of the TtrA subunit of the tetrathionate reductase. In addition, the 3D crystal structure of TtrD is presented at 1.35 ? resolution and a nine-residue binding epitope for TtrD is identified within the TtrA signal peptide close to the twin-arginine targeting motif. This work suggests that archaea may employ a chaperone-dependent Tat proofreading system that is similar to that utilized by bacteria.     

Specific inhibition of the translocation of a subset of Escherichia coli TAT substrates by the TorA signal peptide

The SufI protein and the trimethylamine N-oxide reductase (TorA) are the two best-characterized prototype proteins exported by the Escherichia coli TAT system. Whereas SufI does not contain cofactors, TorA is a molybdo-enzyme and the acquisition of the molybdo-cofactor is a prerequisite for its translocation. The overproduction of each protein leads to the saturation of its translocation, but it was unknown if the overproduction of one substrate could saturate the TAT apparatus and block thus the translocation of other TAT substrates. Here, we showed that the overproduction of SufI saturated only its own translocation, but had no effect of the translocation of TorA and other TAT substrate analyzed. To dissect the saturation mechanism of TorA translocation, we shortened by about one-third of the TorA protein and removed nine consensus molybdo-cofactor-binding ligands. Like SufI, the truncated TorA (TorA502) did not contain cofactor and would not compete with the full length TorA for molybdo-cofactor acquisition. The overproduction of TorA502 completely inhibited the export of the full length TorA and dimethyl sulfoxide (DMSO) reductase, but had no effect on the translocation of SufI, nitrate-induced formate dehydrogenase and hydrogenase-2. Importantly, deletion of the twin-arginine signal peptide of TorA502 abolished the inhibitory effect. Moreover, the overproduction of the TorA signal peptide fused to the green fluorescence protein (GFP) was sufficient to block the TorA translocation. These results demonstrated that the twin-arginine signal peptide of the TorA protein specifically inhibits the translocation of a subset of TAT substrates, probably at the step of their targeting to the TAT apparatus.     

A novel protein fold and extreme domain swapping in the dimeric TorD chaperone from Shewanella massilia

TorD is the cytoplasmic chaperone involved in the maturation of the molybdoenzyme TorA prior to the translocation of the folded protein into the periplasm. The X-ray structure at 2.4 A resolution of the TorD dimer reveals extreme domain swapping between the two subunits. The all-helical architecture of the globular domains within the intertwined molecular dimer shows no similarity with known protein structures. According to sequence similarities, this new fold probably represents the architecture of the chaperones associated with the bacterial DMSO/TMAO reductases and also that of proteins of yet unknown functions. The occurrence of multiple oligomeric forms and the chaperone activity of both monomeric and dimeric TorD raise questions about the possible biological role of domain swapping in this protein.     

Characterization and multiple molecular forms of TorD from Shewanella massilia,the putative chaperone of the molybdoenzyme TorA

Several bacteria use trimethylamine N-oxyde (TMAO) as an exogenous electron acceptor for anaerobic respiration. This metabolic pathway involves expression of the tor operon that codes for a periplasmic molybdopterin-containing reductase of the DMSO/TMAO family, a pentahemic c-type cytochrome, and the TorD cytoplasmic chaperone, possibly required for acquisition of the molybdenum cofactor and translocation of the reductase by the twin-arginine translocation system. In this report, we show that the TorD chaperone from Shewanella massilia forms multiple and stable oligomeric species. The monomeric, dimeric, and trimeric forms were purified to homogeneity and characterized by analytical ultracentrifugation. Small-angle X-ray scattering (SAXS) and preliminary diffraction data indicated that the TorD dimer is made of identical protein modules of similar size to the monomeric species. Interconversion of the native oligomeric forms occurred at acidic pH value. In this condition, ANS fluorescence indicates a non-native conformation of the polypeptide chain in which, according to the circular dichroism spectra, the alpha-helical content is similar to that of the native species. Surface plasmon resonance showed that both the monomeric and dimeric species bind the mature TorA enzyme, but that the dimer binds its target protein more efficiently. The possible biologic significance of these oligomers is discussed in relation to the chaperone activity of TorD, and to the ability of another member of the TorD family to bind the Twin Arginine leader sequences of the precursor of DMSO/TMAO reductases.     

Genetic evidence for a tight cooperation of TatB and TatC during productive recognition of twin-arginine (Tat) signal peptides in Escherichia coli

The twin arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane of bacteria. Tat signal peptides contain a consensus motif (S/T-R-R-X-F-L-K) that is thought to play a crucial role in substrate recognition by the Tat translocase. Replacement of the phenylalanine at the +2 consensus position in the signal peptide of a Tat-specific reporter protein (TorA-MalE) by aspartate blocked export of the corresponding TorA(D(+2))-MalE precursor, indicating that this mutation prevents a productive binding of the TorA(D(+2)) signal peptide to the Tat translocase. Mutations were identified in the extreme amino-terminal regions of TatB and TatC that synergistically suppressed the export defect of TorA(D(+2))-MalE when present in pairwise or triple combinations. The observed synergistic suppression activities were even more pronounced in the restoration of membrane translocation of another export-defective precursor, TorA(KQ)-MalE, in which the conserved twin arginine residues had been replaced by lysine-glutamine. Collectively, these findings indicate that the extreme amino-terminal regions of TatB and TatC cooperate tightly during recognition and productive binding of Tat-dependent precursor proteins and, furthermore, that TatB and TatC are both involved in the formation of a specific signal peptide binding site that reaches out as far as the end of the TatB transmembrane segment.     

The 1.38 A crystal structure of DmsD protein from Salmonella typhimurium, a proofreading chaperone on the Tat pathway

The DmsD protein is necessary for the biogenesis of dimethyl sulphoxide (DMSO) reductase in many prokaryotes. It performs a critical chaperone function initiated through its binding to the twin-arginine signal peptide of DmsA, the catalytic subunit of DMSO reductase. Upon binding to DmsD, DmsA is translocated to the periplasm via the so-called twin-arginine translocation (Tat) pathway. Here we report the 1.38 A crystal structure of the protein DmsD from Salmonella typhimurium and compare it with a close functional homolog, TorD. DmsD has an all-alpha fold structure with a notable helical extension located at its N-terminus with two solvent exposed hydrophobic residues. A major difference between DmsD and TorD is that TorD structure is a domain-swapped dimer, while DmsD exists as a monomer. Nevertheless, these two proteins have a number of common features suggesting they function by using similar mechanisms. A possible signal peptide-binding site is proposed based on structural similarities. Computational analysis was used to identify a potential GTP binding pocket on similar surfaces of DmsD and TorD structures.     

Translocation of green fluorescent protein by comparative analysis with multiple signal peptides

Type I and II secretory pathways are used for the translocation of recombinant proteins from the cytoplasm of Escherichia coli. The purpose of this study was to evaluate four signal peptides (HlyA, TorA, GeneIII, and PelB), representing the most common secretion pathways in E. coli, for their ability to target green fluorescent protein (GFP) for membrane translocation. Signal peptide-GFP genetic fusions were designed in accordance with BioFusion standards (BBF RFC 10, BBF RFC 23). The HlyA signal peptide targeted GFP for secretion to the extracellular media via the type I secretory pathway, whereas TAT-dependent signal peptide TorA and Sec-dependent signal peptide GeneIII exported GFP to the periplasm. The PelB signal peptide was inefficient in translocating GFP. The use of biological technical standards simplified the design and construction of functional signal peptide-recombinant protein genetic devices for type I and II secretion in E. coli. The utility of the standardized parts model is further illustrated as constructed biological parts are available for direct application to other studies on recombinant protein translocation.     

The Escherichia coli TatABC system and a Bacillus subtilis TatAC-type system recognise three distinct targeting determinants in twin-arginine signal peptides

The Tat system transports folded proteins across bacterial and thylakoid membranes. In Gram-negative organisms, it is encoded by tatABC genes and the system recognizes substrates bearing signal peptides with a conserved twin-arginine motif. Most Gram-positive organisms lack a tatB gene, indicating major differences in organisation and/or mechanism. Here, we have characterized the essential targeting determinants that are recognized by a Bacillus subtilis TatAC-type system, TatAdCd. Substitution by lysine of either of the twin-arginine residues in the TorA signal peptide can be tolerated, but the presence of twin-lysine residues blocks export completely. We show that additional determinants can be as important as the twin-arginine motif. Replacement of the −1 serine by alanine, in either the TorA or DmsA signal peptide, almost blocks export by either the B. subtilis TatAdCd or Escherichia coli TatABC systems, firmly establishing the importance of this −1 residue in these signal peptides. Surprisingly, the +2 leucine in the DmsA signal peptide (sequence SRRGLV) appears to play an equally important role and substitution by alanine or phenylalanine blocks export by both the B. subtilis and E. coli systems. These data identify three distinct determinants, whose importance varies depending on the signal peptide in question. The data also show that the B. subtilis TatAdCd and E. coli TatABC systems recognize very similar determinants within their target peptides, and exhibit surprisingly similar responses to mutations within these determinants.