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.     

DmsD is required for the biogenesis of DMSO reductase in Escherichia coli but not for the interaction of the DmsA signal peptide with the Tat apparatus

The DmsD protein is essential for the biogenesis of DMSO reductase in Escherichia coli, and binds the signal peptide of the DmsA subunit, a Tat substrate. This suggests a role as a guidance factor to target pre-DmsA to the translocase. Here, we have analysed the export of fusion proteins in which the DmsA and TorA signal peptides are fused to green fluorescent protein. Both chimeras are efficiently exported to the periplasm in wild-type E. coli cells and we show that their export efficiencies are essentially identical in a mutant lacking DmsD. An authentic Tat substrate, TMAO reductase, is also efficiently exported in the dmsD mutant. The data indicate that DmsD carries out a critical role in DMSO reductase biogenesis/assembly but is not required for the functioning of the DmsA signal peptide.     

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.     

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.     

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 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.     

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.     

‘Come into the fold’: A comparative analysis of bacterial redox enzyme maturation protein members of the NarJ subfamily

Redox enzyme maturation proteins (REMPs) are system-specific chaperones required for the maturation of complex iron sulfur molybdoenzymes that are important for anaerobic respiration in bacteria. Although they perform similar biological roles, REMPs are strikingly different in terms of sequence, structure, systems biology, and type of terminal electron acceptor that it supports for growth. Here we critically dissect current knowledge pertaining to REMPs of the nitrate reductase delta superfamily, specifically recognized in Escherichia coli to include NarJ, NarW, TorD, DmsD, and YcdY, also referred to as the NarJ REMP subfamily. We show that NarJ subfamily members share sequence homology and similar structural features as revealed by alignments performed on structurally characterized REMPs. We include an updated phylogenetic analysis of subfamily members, justifying their classification in this subfamily. The structural and functional roles of each member are presented herein and these discussions suggest that although NarJ subfamily members are related in sequence and structure, each member demonstrates remarkable uniqueness, validating the concept of system-specific chaperones.     

DnaK plays a pivotal role in Tat targeting of CueO and functions beside SlyD as a general Tat signal binding chaperone

The Tat (twin-arginine translocation) system from Escherichia coli transports folded proteins with N-terminal twin-arginine signal peptides across the cytoplasmic membrane. The influence of general chaperones on Tat substrate targeting has not been clarified so far. Here we show that the chaperones SlyD and DnaK bind to a broad range of different Tat signal sequences in vitro and in vivo. Initially, SlyD and GroEL were purified from DnaK-deficient extracts by their affinity to various Tat signal sequences. Of these, only SlyD bound Tat signal sequences also in the presence of DnaK. SlyD and DnaK also co-purified with Tat substrate precursors, demonstrating the binding to Tat signal sequences in vivo. Deletion of dnaK completely abolished Tat-dependent translocation of CueO, but not of DmsA, YcdB, or HiPIP, indicating that DnaK has an essential role specifically for CueO. DnaK was not required for stability of the CueO precursor and thus served in some essential step after folding. A CueO signal sequence fusion to HiPIP was Tat-dependently transported without the need of DnaK, indicating that the mature domain of CueO is responsible for the DnaK dependence. The overall results suggest that SlyD and DnaK are in the set of chaperones that can serve as general Tat signal-binding proteins. DnaK has additional functions that are indispensable for the targeting of CueO.     

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.     

Multiple roles for the twin arginine leader sequence of dimethyl sulfoxide reductase of Escherichia coli

Dimethyl sulfoxide (Me(2)SO) reductase of Escherichia coli is a terminal electron transport chain enzyme that is expressed under anaerobic growth conditions and is required for anaerobic growth with Me(2)SO as the terminal electron acceptor. The trimeric enzyme is composed of a membrane extrinsic catalytic dimer (DmsAB) and a membrane intrinsic anchor (DmsC). The amino terminus of DmsA has a leader sequence with a twin arginine motif that targets DmsAB to the membrane via a novel Sec-independent mechanism termed MTT for membrane targeting and translocation. We demonstrate that the Met-1 present upstream of the twin arginine motif serves as the correct translational start site. The leader is essential for the expression of DmsA, stability of the DmsAB dimer, and membrane targeting of the reductase holoenzyme. Mutation of arginine 17 to aspartate abolished membrane targeting. The reductase was labile in the leader sequence mutants. These mutants failed to support growth on glycerol-Me(2)SO minimal medium. Replacing the DmsA leader with the TorA leader of trimethylamine N-oxide reductase produced a membrane-bound DmsABC with greatly reduced enzyme activity and inefficient anaerobic respiration indicating that the twin arginine leaders may play specific roles in the assembly of redox enzymes.     

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.     

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.     

Sequence-specific binding of prePhoD to soluble TatAd indicates protein-mediated targeting of the Tat export in Bacillus subtilis

The Tat (twin-arginine protein translocation) system initially discovered in the thylakoid membrane of chloroplasts has been described recently for a variety of eubacterial organisms. Although in Escherichia coli four Tat proteins with calculated membrane spanning domains have been demonstrated to mediate Tat-dependent transport, a specific transport system for twin-arginine signal peptide containing phosphodiesterase PhoD of Bacillus subtilis consists of one TatA/TatC (TatAd/TatCd) pair of proteins. Here, we show that TatAd was found beside its membrane-integrated localization in the cytosol were it interacted with prePhoD. prePhoD was efficiently co-immunoprecipitated by TatAd. Inefficient co-immunoprecipitation of mature PhoD and missing interaction to Sec-dependent and cytosolic peptides by TatAd demonstrated a particular role of the twin-arginine signal peptide for this interaction. Affinity of prePhoD to TatAd was interfered by peptides containing the twin-arginine motif but remained active when the arginine residues were substituted. The selective binding of TatAd to peptides derived from the signal peptide of PhoD elucidated the function of the twin-arginine motif as a target site for pre-protein TatAd interaction. Substitution of the binding motif demonstrated the pivotal role of basic amino acid residues for TatA binding. These features suggest that TatA interacts prior to membrane integration with its pre-protein substrate and could therefore assist targeting of twin-arginine pre-proteins.     

Essential cytoplasmic domains in the Escherichia coli TatC protein.

The twin-arginine translocation (Tat) system mediates the transport of proteins across the bacterial plasma membrane and chloroplast thylakoid membrane. Operating in parallel with Sec-type systems in these membranes, the Tat system is completely different in both structural and mechanistic terms, and is uniquely able to catalyze the translocation of fully folded proteins across coupled membranes. TatC is an essential, multispanning component that has been proposed to form part of the binding site for substrate precursor proteins. In this study we have tested the importance of conserved residues on the periplasmic and cytoplasmic face of the Escherichia coli protein. We find that many of the mutations on the cytoplasmic face have little or no effect. However, substitution at several positions in the extreme N-terminal cytoplasmic region or the predicted first cytoplasmic loop lead to a significant or complete loss of Tat-dependent export. The mutated strains are unable to grow anaerobically on trimethylamine N-oxide minimal media and are unable to export trimethylamine-N-oxide reductase (TorA). The same mutants are completely unable to export a chimeric protein, comprising the TorA signal peptide linked to green fluorescent protein, indicating that translocation is blocked rather than cofactor insertion into the TorA mature protein. The data point to two essential cytoplasmic domains on the TatC protein that are essential for export.     

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.     

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.     

Signal peptidase I-mediated processing of an engineered mammalian cytochrome b(5) precursor is an exocytoplasmic post-translocational event in Escherichia coli

Proteins destined for translocation across the prokaryotic cytoplasmic membrane are synthesized as precursors carrying transient N-terminal extensions known as signal sequences. They facilitate initial engagement of precursor proteins with the sec-dependent translocase to initiate active threading of the polypeptide across the membrane. The translocated precursor is then processed by a transcytoplasmic signal peptidase anchored to the inner membrane. The temporal nature of cleavage of the signal sequence during pre-protein translocation has remained elusive. Using an engineered mammalian cytochrome b(5) precursor we demonstrate that the signal peptide processing in Escherichia coli is an event that can occur after almost complete exocytoplasmic translocation of the preprotein is accomplished. We discuss implications of the findings in light of the known working model of sec-dependent pre-protein translocon.     

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.     

Signal peptidase I-mediated processing of an engineered mammalian cytochrome b5 precursor is an exocytoplasmic post-translocational event in Escherichia coli

Proteins destined for translocation across the prokaryotic cytoplasmic membrane are synthesized as precursors carrying transient N-terminal extensions known as signal sequences. They facilitate initial engagement of precursor proteins with the sec-dependent translocase to initiate active threading of the polypeptide across the membrane. The translocated precursor is then processed by a transcytoplasmic signal peptidase anchored to the inner membrane. The temporal nature of cleavage of the signal sequence during pre-protein translocation has remained elusive. Using an engineered mammalian cytochrome b₅ precursor we demonstrate that the signal peptide processing in Escherichia coli is an event that can occur after almost complete exocytoplasmic translocation of the preprotein is accomplished. We discuss implications of the findings in light of the known working model of sec-dependent pre-protein translocon.