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.     

Identification of residues in DmsD for twin-arginine leader peptide binding, defined through random and bioinformatics-directed mutagenesis

The twin-arginine translocase (Tat) system is used for the targeting and translocation of folded proteins across the cell membrane of most bacteria. Substrates of this system contain a conserved "twin-arginine" (RR) motif within their signal/leader peptide sequence. Many Tat substrates have their own system-specific chaperone called redox enzyme maturation proteins (REMPs). Here, we study the binding of DmsD, the REMP for dimethyl sulfoxide reductase in Escherichia coli, toward the RR-containing leader peptide of the catalytic subunit DmsA. We have used a multipronged approach targeted at the amino acid sequence of DmsD to define residues and regions important for recognition of the DmsA leader sequence. Residues identified through bioinformatics and THEMATICS analysis were mutated using site-directed mutagenesis. These DmsD residue variants were purified and screened with an in vitro dot-blot far-Western assay to analyze the binding to the DmsA leader sequence. Degenerative polymerase chain reaction was also used to produce a bank of random DmsD amino acid mutants, which were then screened by an in vivo bacterial two-hybrid assay. Using this hybrid method, each DmsD variant was classified into one of three groups based on their degree of interaction with the DmsA leader (none, weak, and moderate). The data from both the in vitro and in vivo analyses were then applied to a model structure of DmsD based on the crystal structure of the Salmonella typhimurium homologue. Our results illustrate the positions of important DmsD residues involved in binding the DmsA leader peptide and identify a "hot pocket" of residues important for leader binding on the structure of DmsD.     

Purification of a Tat leader peptide by co-expression with its chaperone

We present a method for the purification of the 45 residue long leader peptide of Escherichia coli dimethyl sulfoxide reductase subunit A (DmsA(L)), a substrate of the twin arginine translocase, by co-expressing the leader peptide with its specific chaperone protein, DmsD. The peptide can be isolated from the soluble DmsA(L)/DmsD complex or conveniently from the lysate pellet fraction. The recombinant leader peptide is functionally intact as the peptide/chaperone complex can be reconstituted from purified DmsA(L) and DmsD. A construct with DmsA(L) fused to the N-terminus of DmsD (DmsA(L)-DmsD fusion) was created to further explore the properties of the leader peptide-chaperone interactions. Analytical size-exclusion chromatography in-line with multi-angle light scattering reveals that the DmsA(L)-DmsD fusion construct forms a dimer wherein each protomer binds the neighboring leader peptide. A model of this homodimeric interaction is presented.     

Visualizing Interactions along the Escherichia coli Twin-Arginine Translocation Pathway Using Protein Fragment Complementation

The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways.     

Physical nature of signal peptide binding to DmsD

Here we describe the biophysical characterization of the interaction of the redox enzyme maturation protein DmsD with the signal peptide of its target protein, DmsA. Isothermal titration calorimetry (ITC), size exclusion chromatography (SEC), and an in vitro Far-Western assay is used to show that DmsD binds the twin-arginine signal peptide from DmsA in the micromolar range and in a 1:1 molar ratio. The SEC also shows that there is no oligomerization upon binding. Urea and guanidium hydrochloride denaturation profiles demonstrate the stability of DmsD and give insights on how electrostatic and hydrophobic interactions are important within this binding process. Furthermore, by use of N- and C-terminal fusions of DmsA signal peptide to GST, we observe that N-terminal display of the peptide is important for binding DmsD. In addition, all the folding forms of DmsD were found to bind the DmsA signal peptide as observed with the Far-Western assay.     

DmsD,a Tat system specific chaperone,interacts with other general chaperones and proteins involved in the molybdenum cofactor biosynthesis

Many bacterial oxidoreductases depend on the Tat translocase for correct cell localization. Substrates for the Tat translocase possess twin-arginine leaders. System specific chaperones or redox enzyme maturation proteins (REMPs) are a group of proteins implicated in oxidoreductase maturation. DmsD is a REMP discovered in Escherichia coli, which interacts with the twin-arginine leader sequence of DmsA, the catalytic subunit of DMSO reductase. In this study, we identified several potential interacting partners of DmsD by using several in vitro protein–protein interaction screening approaches, including affinity chromatography, co-precipitation, and cross-linking. Candidate hits from these in vitro findings were analyzed by in vivo methods of bacterial two-hybrid (BACTH) and bimolecular fluorescence complementation (BiFC). From these data, DmsD was confirmed to interact with the general molecular chaperones DnaK, DnaJ, GrpE, GroEL, Tig and Ef-Tu. In addition, DmsD was also found to interact with proteins involved in the molybdenum cofactor biosynthesis pathway. Our data suggests that DmsD may play a role as a “node” in escorting its substrate through a cascade of chaperone assisted protein-folding maturation events.     

Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli

The twin-arginine translocation (Tat) system targets cofactor-containing proteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin-arginine motif. In this study, we have analysed the mechanism and capabilities of the E. coli Tat system using green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active. Export is almost totally blocked in tat deletion mutants, indicating that the observed export in wild-type cells occurs predominantly, if not exclusively, by the Tat pathway. Imaging studies reveal a halo of fluorescence in wild-type cells corresponding to the exported periplasmic form; the GFP is distributed uniformly throughout the cytoplasm in a tat mutant. Because previous work has shown GFP to be incapable of folding in the periplasm, we propose that GFP is exported in a fully folded, active state. These data also show for the first time that heterologous proteins can be exported in an active form by the Tat pathway.     

Identification of a twin-arginine leader-binding protein

The transport and targeting of a number of periplasmic proteins is carried out by the Sec-independent Mtt (also referred to as Tat) protein translocase. Proteins using this translocase have a distinct twin-arginine-containing leader. We hypothesized that specific leader-binding proteins exist to escort proteins to the translocase complex. A fusion was constructed with the twin-arginine leader from dimethyl sulphoxide (DMSO) reductase, subunit DmsA, to the N-terminus of glutathione-S-transferase. This leader fusion was bound to a glutathione affinity column through which an Escherichia coli anaerobic cell-free extract was passed. Proteins that bound to the leader were then separated and identified by N-terminal sequencing, which identified DnaK and a protein originating from the uncharacterized reading frame ynfI. This gene has been designated dmsD based on the findings presented in this paper. DmsD was purified as a His6 fusion and was shown to interact with preprotein forms of DmsA and TorA (trimethyl amine N-oxide reductase). A strain carrying a dmsD knock-out mutation showed a loss of anaerobic growth on glycerol-DMSO medium and reduced growth on glycerol-fumarate medium. This work suggests that DmsD is a twin-arginine leader-binding protein.     

The twin-arginine leader-binding protein,DmsD, interacts with the TatB and TatC subunits of the Escherichia coli twin-arginine translocase

The twin-arginine translocase (Tat) pathway is involved in the targeting and translocation of fully folded proteins to the inner membrane and periplasm of bacteria. Proteins that use this pathway contain a characteristic twin-arginine signal sequence, which interacts with the receptor complex formed by the TatBC subunits. Recently, the DmsD protein was discovered, which binds to the twin-arginine signal sequences of the anaerobic respiratory enzymes dimethylsulfoxide reductase (DmsABC) and trimethylamine N-oxide (TMAO) reductase. In this work, the targeting of DmsD within Escherichia coli was investigated. Using cell fractionation and Western blot analysis, DmsD is found to be associated with the inner membrane of wild-type E. coli and a dmsABC mutant E. coli under anaerobic conditions. In contrast, DmsD is predominantly found in the cytoplasmic fraction of a Delta tatABCDE strain, which suggests that DmsD interacts with the membrane-associated Tat complex. Under aerobic conditions DmsD was also found primarily in the cytoplasmic fraction of wild-type E. coli, suggesting that physiological conditions have a significant effect upon the targeting of DmsD to the inner membrane. Size exclusion chromatography data and membrane washing studies indicate that DmsD is interacting tightly with an integral membrane protein and not with the lipid component of the E. coli inner membrane. Additional investigation into the nature of this interaction revealed that the TatB and TatC subunits of the translocase are important for the interaction of DmsD with the E. coli inner membrane.     

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.     

Sequence analysis of bacterial redox enzyme maturation proteins (REMPs)

The twin-arginine protein transport (Tat) system is a remarkable molecular machine dedicated to the translocation of fully folded proteins across energy-transducing membranes. Complex cofactor-containing Tat substrates acquire their cofactors prior to export, and substrate proteins actually require to be folded before transport can proceed. Thus, it is very likely that mechanisms exist to prevent wasteful export of immature Tat substrates or to curb competition between immature and mature substrates for the transporter. Here we assess the primary sequence relationships between the accessory proteins implicated in this process during assembly of key respiratory enzymes in the model prokaryote Escherichia coli. For each respiratory enzyme studied, a redox enzyme maturation protein (REMP) was assigned. The main finding from this review was the hitherto unexpected link between the Tat-linked REMP DmsD and the nitrate reductase biosynthetic protein NarJ. The evolutionary link between Tat transport and cofactor insertion processes is discussed.     

Specificity of signal peptide recognition in tat-dependent bacterial protein translocation

The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) of Zymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host Escherichia coli, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic E. coli Tat signal peptide, which is derived from pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the E. coli periplasm. This export was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sodium azide, and was blocked in E. coli tatC and tatAE mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.     

The twin arginine consensus motif of Tat signal peptides is involved in Sec-independent protein targeting in Escherichia coli

In Escherichia coli a subset of periplasmic proteins is exported through the Tat pathway to which substrates are directed by an NH(2)-terminal signal peptide containing a consensus SRRXFLK "twin arginine" motif. The importance of the individual amino acids of the consensus motif for in vivo Tat transport has been assessed by site-directed mutagenesis of the signal peptide of the Tat substrate pre-SufI. Although the invariant arginine residues are crucial for efficient export, we find that slow transport of SufI is still possible if a single arginine is conservatively substituted by a lysine residue. Thus, in at least one signal peptide context there is no absolute dependence of Tat transport on the arginine pair. The consensus phenylalanine residue was found to be a critical determinant for efficient export but could be functionally substituted by leucine, another amino acid with a highly hydrophobic side chain. Unexpectedly, the consensus lysine residue was found to retard Tat transport. These observations and others suggest that the sequence conservation of the Tat consensus motif is a reflection of the functional importance of the consensus residues. Tat signal peptides characteristically have positively charged carboxyl-terminal regions. However, changing the sign of this charge does not affect export of SufI.     

1H, 13C and 15N resonance assignments and peptide binding site chemical shift perturbation mapping for the Escherichia coli redox enzyme chaperone DmsD

Herein are reported the mainchain ¹H, ¹³C and ¹⁵N chemical shift assignments and amide ¹⁵N relaxation data for Escherichia coli DmsD, a 23.3 kDa protein responsible for the correct folding and translocation of the dimethyl sulfoxide reductase enzyme complex. In addition, the observed amide chemical shift perturbations resulting from complex formation with the reductase subunit DmsA leader peptide support a model in which the 44 residue peptide makes extensive contacts across the surface of the DmsD protein.     

Escherichia coli twin arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities

The twin arginine (Tat) secretion pathway allows the translocation of folded proteins across the cytoplasmic membrane of bacteria. Tat-specific signal peptides contain a characteristic amino acid motif ((S/T)RRXFLK) including two highly conserved consecutive arginine residues that are thought to be involved in the recognition of the signal peptides by the Tat translocase. Here, we have analyzed the specificity of Tat signal peptide recognition by using a genetic approach. Replacement of the two arginine residues in a Tat-specific precursor protein by lysine-glutamine resulted in an export-defective mutant precursor that was no longer accepted by the wild-type translocase. Selection for restored export allowed for the isolation of Tat translocases possessing single mutations in either the amino-terminal domain of TatB or the first cytosolic domain of TatC. The mutant Tat translocases still efficiently accepted the unaltered precursor protein, indicating that the substrate specificity of the translocases was not strictly changed; rather, the translocases showed an increased tolerance toward variations of the amino acids occupying the positions of the twin arginine residues in the consensus motif of a Tat signal peptide.     

The superoxide dismutase SodA is targeted to the periplasm in a SecA-dependent manner by a novel mechanism

The manganese/iron-type superoxide dismutase (SodA) of Rhizobium leguminosarum bv. viciae 3841 is exported to the periplasm of R. l. bv. viciae and Escherichia coli. However, it does not possess a hydrophobic cleaved N-terminal signal peptide typically present in soluble proteins exported by the Sec-dependent (Sec) pathway or the twin-arginine translocation (TAT) pathway. A tatC mutant of R. l. bv. viciae exported SodA to the periplasm, ruling out export of SodA as a complex with a TAT substrate as a chaperone. The export of SodA was unaffected in a secB mutant of E. coli, but its export from R. l. bv. viciae was inhibited by azide, an inhibitor of SecA ATPase activity. A temperature-sensitive secA mutant of E. coli was strongly reduced for SodA export. The 10 N-terminal amino acid residues of SodA were sufficient to target the reporter protein alkaline phosphatase to the periplasm. Our results demonstrate the export of a protein lacking a classical signal peptide to the periplasm by a SecA-dependent, but SecB-independent targeting mechanism. Export of the R. l. bv. viciae SodA to the periplasm was not limited to the genus Rhizobium, but was also observed in other proteobacteria.     

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.     

Export of Thermus thermophilus cytoplasmic beta-glycosidase via the E. coli Tat pathway

The Tat pathway is distinct from the Sec machinery given its unusual capacity to export folded proteins, which contain a twin-arginine (RR) signal peptide, across the plasma membrane. The functionality of the Tat pathway has been demonstrated for several Gram-negative and Gram-positive mesophilic bacteria. To assess the specificity of the Tat system, and to analyze the capacity of a mesophilic bacterial Tat system to translocate cytoplasmic proteins from hyperthermophilic bacteria, we fused the Thermus thermophilus beta-glycosidase (Glc) to the twin-arginine signal peptide of the E. coli TorA protein. When expressed in E. coli, the thermophilic RR-Glc chimera was successfully synthesized and efficiently translocated into the periplasm of the wild type strain. In contrast, the beta-glycosidase accumulated within the cytoplasm of all the tat mutants analyzed. The beta-glycosidase synthesized in these strains exhibited thermophilic properties. These results demonstrated, for the first time, the capacity of the E. coli Tat system to export cytoplasmic hyperthermophilic protein, implying an important potential of the Tat system for the production of thermostable enzymes used in bioprocessing applications.     

An essential role for the DnaK molecular chaperone in stabilizing over-expressed substrate proteins of the bacterial twin-arginine translocation pathway

All secreted proteins in Escherichia coli must be maintained in an export-competent state before translocation across the inner membrane. In the case of the Sec pathway, this function is carried out by the dedicated SecB chaperone and the general chaperones DnaK-DnaJ-GrpE and GroEL-GroES, whose job collectively is to render substrate proteins partially or entirely unfolded before engagement of the translocon. To determine whether these or other general molecular chaperones are similarly involved in the translocation of folded proteins through the twin-arginine translocation (Tat) system, we screened a collection of E. coli mutant strains for their ability to transport a green fluorescent protein (GFP) reporter through the Tat pathway. We found that the molecular chaperone DnaK was essential for cytoplasmic stability of GFP bearing an N-terminal Tat signal peptide, as well as for numerous other recombinantly expressed endogenous and heterologous Tat substrates. Interestingly, the stability conferred by DnaK did not require a fully functional Tat signal as substrates bearing translocation defective twin lysine substitutions in the consensus Tat motif were equally unstable in the absence of DnaK. These findings were corroborated by crosslinking experiments that revealed an in vivo association between DnaK and a truncated version of the Tat substrate trimethylamine N-oxide reductase (TorA502) bearing an RR or a KK signal peptide. Since TorA502 lacks nine molybdo-cofactor ligands essential for cofactor attachment, the involvement of DnaK is apparently independent of cofactor acquisition. Finally, we show that the stabilizing effects of DnaK can be exploited to increase the expression and translocation of Tat substrates under conditions where the substrate production level exceeds the capacity of the Tat translocase. This latter observation is expected to have important consequences for the use of the Tat system in biotechnology applications where high levels of periplasmic expression are desirable.