Intrinsic GTPase activity of a bacterial twin-arginine translocation proofreading chaperone induced by domain swapping David Guymer1, Julien Maillard2, Mark F Agacan1, Charles A Brearley3 and Frank Sargent1 College of Life Sciences, University of Dundee, Dundee, UK ENAC-ISTE ⁄ Laboratoire de Biotechnologie Environnementale (LBE), EPF Lausanne, Switzerland School of Biological Sciences, University of East Anglia, Norwich, UK Keywords Escherichia coli; molecular chaperones; noncanonical GTPase; TorD protein; twin-arginine transport pathway Correspondence F Sargent, Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK Fax: +44 1382 388 216 Tel: +44 1382 386 463 E-mail: f.sargent@dundee.ac.uk (Received 28 July 2009, revised 12 November 2009, accepted 18 November 2009) doi:10.1111/j.1742-4658.2009.07507.x The bacterial twin-arginine translocation (Tat) system is a protein targeting pathway dedicated to the transport of folded proteins across the cytoplasmic membrane Proteins transported on the Tat pathway are synthesised as precursors with N-terminal signal peptides containing a conserved amino acid motif In Escherichia coli, many Tat substrates contain prosthetic groups and undergo cytoplasmic assembly processes prior to the translocation event A pre-export ‘Tat proofreading’ process, mediated by signal peptide-binding chaperones, is considered to prevent premature export of some Tat-targeted proteins until all other assembly processes are complete TorD is a paradigm Tat proofreading chaperone and co-ordinates the maturation and export of the periplasmic respiratory enzyme trimethylamine N-oxide reductase (TorA) Although it is well established that TorD binds directly to the TorA signal peptide, the mechanism of regulation or control of binding is not understood Previous structural analyses of TorD homologues showed that these proteins can exist as monomeric and domainswapped dimeric forms In the present study, we demonstrate that isolated recombinant TorD exhibits a magnesium-dependent GTP hydrolytic activity, despite the absence of classical nucleotide-binding motifs in the protein TorD GTPase activity is shown to be present only in the domain-swapped homodimeric form of the protein, thus defining a biochemical role for the oligomerisation Site-directed mutagenesis identified one TorD side-chain (D68) that was important in substrate selectivity A D68W variant TorD protein was found to exhibit an ATPase activity not observed for native TorD, and an in vivo assay established that this variant was defective in the Tat proofreading process Structured digital abstract l MINT-7302371, MINT-7302377: TorD (uniprotkb:P36662) and TorD (uniprotkb:P36662) bind (MI:0407) by molecular sieving (MI:0071) l MINT-7302402: TorD (uniprotkb:P36662) and TorD (uniprotkb:P36662) bind (MI:0407) by comigration in non denaturing gel electrophoresis (MI:0404) l MINT-7302387: TorD (uniprotkb:P36662) and TorD (uniprotkb:P36662) bind (MI:0407) by cosedimentation in solution (MI:0028) Abbreviations GAP, GTPase activating protein; IMAC, immobilised metal affinity chromatography; MGD, molybdopterin guanine dinucleotide; Tat, twin-arginine translocation; TMAO, trimethylamine N-oxide; TorA, trimethylamine N-oxide reductase FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS 511 The E coli TorD homodimer has GTPase activity D Guymer et al Introduction The twin-arginine translocation (Tat) system is a protein-targeting pathway present in the cytoplasmic membrane of many prokaryotes [1] Tat-targeted proteins are synthesised as precursors with cleavable N-terminal signal peptides incorporating the distinctive SRRxFLK ‘twin-arginine’ amino acid motif [2] A key feature of Tat translocation is the requirement for physiological substrates to be fully folded before successful translocation can occur [3] Escherichia coli produces 27 Tat substrates [4], the majority of which bind complex prosthetic groups, fold, activate, and often oligomerise, in the cytoplasm prior to membrane translocation [1,3,5] It is considered that the Tat translocase itself may be able to accept or reject pre-proteins on the basis of their folded state in a ‘Tat quality control’ process [3] In addition, some proteins are subjected to a chaperone-mediated ‘Tat proofreading’ process in the cytoplasm prior to export Tat proofreading involves the direct binding of particular Tat signal peptides by dedicated chaperones aiming to prevent premature targeting of immature proteins [6,7] The Escherichia coli trimethylamine N-oxide (TMAO) reductase (TorA) is a Tat-dependent periplasmic redox enzyme encoded by the torCAD operon [8] TorA is the archetypal model Tat substrate synthesised with a 39 residue signal peptide and contains molybdopterin guanine dinucleotide (MGD) as a single prosthetic group [9] Acquisition of the MGD cofactor by TorA in the cell cytoplasm is an essential pre-requisite to translocation of this protein [10] The torD gene product is involved in cofactor loading into TorA [11–18], and additionally operates as a Tat proofreading chaperone, binding directly to the TorA twin-arginine signal peptide [5,19,20] TorD belongs to a family of peptide-binding proteins specifically dedicated to molybdoprotein assembly [6,7,16,21] Phylogenetic analysis of the TorD family allows separation of the members into three broad ‘clades’: the TorD clade, the DmsD clade and the NarJ clade [16,21] A number of 3D structures now exist for members of the TorD family and these all share a unique all a-helical architecture [22–25] The helices are arranged into two domains (N-terminal and C-terminal), which are connected by a hinge region [23] Crystal structures of monomeric forms have been described [22,24,25]; however, higher-order oligomers of TorD-like proteins have been observed [26,27] and the crystal structure of a homodimer has been solved [23] Dimerisation of the Shewanella massilia TorD protein is driven by ‘domain-swapping’ in which the N-terminal domain of one protomer packs 512 onto the C-terminal domain of a second protomer (and vice versa); however, the physiological role of this domain-swapping was not clear [23] The isolated, recombinant E coli TorD monomer has been shown to bind to the TorA signal peptide in vitro with an apparent dissociation constant (Kd) of 59 nm [19] Such relatively tight binding led to some speculation as to how binding and release cycles could be regulated The crystal structure of the Sh massilia TorD homodimer was observed to contain tightlybound oxidised (and therefore cyclic) dithiothreitol [23] As a result, Hatzixanthis et al [20] hypothesised that this observation could suggest that a common cyclic regulatory molecule, perhaps a nucleotide, could normally be bound by TorD Indeed, the monomeric form of the E coli TorD protein was subsequently shown to bind guanine nucleotides with low affinity (apparent Kd  370 lm for GTP) [20], and recent independent computational analysis predicted a potential GTP-binding site on the DmsD protein from Salmonella enterica serovar Typhimurium [24] In the present study, the relationship between E coli TorD and GTP was investigated We demonstrate that recombinant TorD possesses an intrinsic magnesiumdependent GTP hydrolysis activity in vitro It is revealed that this activity is a property only of the dimeric form of the protein, suggesting that domainswapping is required to generate the active site In addition, site-directed mutagenesis identifies one residue, D-68, that is involved in the substrate selectivity of this protein Results TorD has magnesium-dependent GTPase activity Early ligand-binding experiments [20] and recent bioinformatic analysis [24] suggested that TorD-like proteins may bind GTP In addition, overproduction of TorD family proteins has been reported to result in the isolation of a range of stable homo-oligomeric forms [26,27] In the initial development of our purification strategies, we established that an ion exchange chromatographic step, immediately following metal affinity chromatography, resulted in isolation of stable monomeric TorDhis [20,28] This monomeric form was ideal for biophysical studies [19,20] but, despite its ability to bind guanine nucleotides, no nucleotide hydrolysis activity could be detected [20] We therefore decided to explore the possibility that different folding forms of TorDhis may harbour different biological activities FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS D Guymer et al First, E coli TorDhis was overproduced and isolated by nickel-affinity chromatography Eluate from the metal affinity column was then assayed directly for GTP and ATP hydrolytic activity using a malachite green method for the quantification of free inorganic phosphate (Pi) This assay measures free Pi in solution by spectrophotometric determination of the complex formed between malachite green, molybdate and free Pi The initial assay chosen already included magnesium chloride in the reaction mixture because magnesium ions are essential for the GTPase activity of the majority of characterised GTPases [29,30] The assay demonstrated that TorD exhibits hydrolytic activity towards GTP (Fig 1A) No Pi release was detected in the negative controls, which included a sample of the elution buffer used in the chromatographic experiment and a sample of a maltose-binding protein isolated in the identical buffers, and on the same immobilised metal affinity chromatography (IMAC) column, as the TorDhis described here (Fig 1A) In addition, our recombinant TorDhis shows no hydrolytic activity towards ATP (Fig 1A) The binding of a magnesium cofactor has long been established as essential for the activity of canonical GTP-binding proteins [29,31] The precise role of Mg2+ may vary between enzymes but is clearly essential for GTP hydrolysis and is almost always required to allow GTP binding [29,30] To determine the requirement for magnesium of the GTPase activity of TorD, the reaction was performed in varying amounts of MgCl2 (Fig 1B) The GTPase activity of TorD without added MgCl2 is negligible, establishing that magnesium is required for TorD GTPase activity (Fig 1B) Titration of increasing amounts of MgCl2 gradually enhances GTPase activity, with a peak at approximately mm (Fig 1B) The malachite green assay was also performed with 1.2 mm MgCl2 in the presence of 10 mm EDTA In this experiment, the presence of EDTA completely inhibited the reaction (data not shown) Furthermore, no other divalent cations, including manganese, could replace magnesium in this assay (not shown) Finally, the product of the GTP hydrolysis reaction catalysed by TorD was shown to be GDP by HPLC analysis (Fig S1) Taken altogether, these data demonstrate the initial identification of a strictly magnesiumdependent GTPase activity associated with the IMAC pool of recombinant TorD protein GTP hydrolytic activity is a feature of the TorD homodimer Having established that GTPase activity was associated with TorD collected immediately following metal The E coli TorD homodimer has GTPase activity Fig TorD has magnesium-dependent GTPase activity (A) The malachite green assay for in vitro Pi release from nucleotides Reaction mixtures [50 lL aliquots containing mM GTP or ATP, 1.2 mM MgCl2 and 10 mM Tris–HCl (pH 7.5)] were incubated at 22 °C for 24 h containing 0.1 mM of metal affinity chromatographypurified TorD (‘TorD + GTP’ and ‘TorD + ATP’) As controls, an equal amount of a His-tagged maltose binding protein-TorA signal peptide fusion (‘MalE:TorA-SP + GTP’), or protein-free column buffer (‘no protein’), were also assayed in the presence of mM GTP (B) The GTP hydrolysis reaction is magnesium dependent Reactions containing 0.1 mM of metal affinity chromatography-purified TorD with mM GTP buffered in 10 mM Tris–HCl (pH 7.5) were incubated at 22 °C for 24 h in the presence of increasing amounts of MgCl2 Released Pi was assayed by the malachite green method In both (A) and (B), the total phosphate released in each reaction is shown and error bars represent the SEM (n = 3) affinity chromatography, the next step was to further purify the hydrolytic activity by alternative chromatographic techniques Size exclusion chromatography using a SuperdexÔ 75 column identified a range of molecular mass species present in the nickel-affinity purified TorD sample (Fig 2A) The major peak corresponded to an approximate molecular mass of 25.5 kDa, in agreement with the predicted molecular mass of TorDhis of 24.2 kDa, and so likely represents monomeric TorD Lesser protein peaks representing TorD species with approximate molecular mass of FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS 513 The E coli TorD homodimer has GTPase activity A B C D Guymer et al 49.0 kDa, similar to the predicted 48.5 kDa of a dimer of TorDhis, and higher-order oligomers at approximately 85.7 kDa (beyond the linear range of the SuperdexÔ 75 column of 3–70 kDa) were also observed (Fig 2A) The presence of TorD oligomers is supported by gel electrophoresis (Fig 2B, C) Denaturing SDS–PAGE showed the presence of TorD polypeptide in all fractions tested, whereas PAGE performed in the absence of SDS allowed a ready visualisation of the different oligomeric forms of TorD present in the higher molecular mass fractions (Fig 2B, C) Magnesiumdependent GTPase activity was restricted to the higher molecular mass fractions, and was completely absent from the monomer form (data not shown) CibacronÔ Blue F3G-A is a dye molecule that can be immobilised to a Sepharose matrix (Blue SepharoseÔ HP), and which is able to bind specifically to some nucleotide-binding proteins as a result of its structural similarity to nucleotide cofactors Specifically-bound proteins are then normally eluted by the application of either an amount of cofactor or by increasing the ionic strength Initial experiments with TorD under ‘standard’ conditions for analysing classical nucleotide-binding proteins [32] suggested that the protein did not bind tightly to Cibacron Blue under conditions of low ionic strength, and that nothing was therefore eluted upon application of a high ionic strength solution (data not shown) Surprisingly, however, upon washing the Blue Sepharose column in water after each experiment, a small peak of protein was observed in the eluate To explore this further, the buffer conditions were changed and the metal affinity pool of TorD was applied to a Blue Sepharose column equilibrated in buffer containing 0.5 m NaCl The elution profile revealed that the majority of the sample did not bind to the CibacronÔ Blue dye (Fig 3A) However, upon switching from the relatively high salt Fig Oligomeric forms of E coli TorD can be readily identified (A) Elution profile of a pool of TorD derived from metal affinity chromatography applied to a SuperdexÔ 75 (10 ⁄ 30) size exclusion column at 0.5 mLỈmin)1 in 50 mM Tris–HCl (pH 7.5) and 200 mM NaCl Values for the apparent molecular mass of peak proteins were calculated using control proteins of known molecular mass (not shown) (B) Equivalent volumes (5 lL) of the protein fractions indicated were diluted : in either Laemmli or native sample buffer and subjected directly (unboiled) to SDS–PAGE (top panel) and nonSDS–PAGE (bottom panel) on 12.5% (w ⁄ v) polyacrylamide gels Protein bands were visualised with Coomassie Brilliant Blue R-250 stain (C) Equivalent amounts (3.6 lg) of protein in each indicated fraction were separated directly (unboiled) by SDS–PAGE (top panel) and non-SDS–PAGE (bottom panel) on 12.5% (w ⁄ v) polyacrylamide gels and stained with Coomassie R-250 514 FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS D Guymer et al Absorbance units (280 nm) 1400 60 1200 50 1000 40 800 30 600 20 400 10 200 12 Conductance units (mS·cm–1) A The E coli TorD homodimer has GTPase activity 17 22 27 Ve (ml) 32 37 B C D E concentration buffer (0.5 m) to a very low ionic strength solution (pure water in this case), a small peak of protein was eluted from the column (Fig 3A) SDS–PAGE and western analysis revealed that this fraction contained TorD protein (Fig 3B, C), and TorD isolated in this way is referred to in the present study as ‘TorDBlue’ The TorDBlue protein peak isolated by the ‘reverse’ Blue Sepharose chromatography protocol was analysed Fig TorD GTPase activity can be isolated by Cibacron Blue affinity chromatography (A) Unusual behaviour of TorD on CibacronÔ Blue affinity media A sample of 0.5 mM metal affinity chromatography-purified TorD was applied to a mL HiTrapÔ Blue column, attached to an FPLC system, in 0.5 M NaCl-containing buffer at mLỈmin)1 Bound proteins were eluted in a single step to pure water (B) Protein fractions from the unbound flow-through (‘FT’) or single mL fractions eluted in water (numbered 35–38) were diluted : in either Laemmli or ‘native’ sample buffer and subjected (unboiled) to SDS–PAGE (top panel) and non-SDS–PAGE (bottom panel) Three microlitres of the flow-through sample was used to give an equivalent amount of protein loaded compared to that of 36 mL fraction ( 1.1 lg) Gels were stained with Coomassie R-250 (C) A western immunoblot was carried out on ng samples of the original metal affinity-purified material (‘IMAC’), the unbound flow-through (‘FT’), and pooled fractions 36–38 Proteins were mixed with Laemmli disaggregation buffer and heat-treated at 100 °C for before being separated by SDS–PAGE, blotted onto nitrocellulose, and challenged with an anti-TorD serum (1 : 10 000 dilution) (D) Analysis of the ‘TorDBlue’ fractions for GTPase activity Protein samples (1.2 lg) of fractions eluted in water from the Cibacron Blue Sepharose column were subjected to the malachite green GTPase assay The fraction at 33 mL contained no detectable protein and was assayed as an internal control to establish that incubation with the column buffers alone did not facilitate Pi release Error bars indicate the SEM (n = 3) (E) Pooled protein samples (4 lg) from the original metal affinity column (‘IMAC’) and the combined eluate from the CibacronÔ Blue affinity column (‘Blue’) were mixed with Laemmli disaggregation buffer and heattreated at 100 °C for before being separated by SDS–PAGE using Bio-Rad (Bio-Rad, Hercules, CA, USA) pre-cast 15% (w ⁄ v) polyacrylamide gels Proteins were visualised using the SilverquestÒ (Invitrogen) silver-staining kit and, where indicated, the reaction was stopped after or for GTPase activity using the malachite green assay (Fig 3D) Very interestingly, TorDBlue demonstrated GTPase activity, whereas the ‘flow-through’ fraction of TorD showed negligible activity (Fig 3D) Control experiments using a column fraction containing no protein also showed no activity (Fig 3D) Very unusually, therefore, all of the GTPase activity was bound to the Cibacron Blue column in the presence of 0.5 m salt (something that a ‘canonical’ nucleotide-binding protein would not do) and, subsequently, all of the GTPase activity could be eluted in a solution of very low ionic strength (again not the typical behaviour of a classical nucleotide-binding protein) The relative purity of the TorDBlue preparation was analysed further by SDS–PAGE and MS Both the TorDhis fraction (from the initial metal affinity chromatography pool) and the TorDBlue preparation were separated by SDS–PAGE and the proteins were visualised using the most sensitive silver staining method available (Fig 3E) This method revealed a single FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS 515 The E coli TorD homodimer has GTPase activity D Guymer et al strong band in each fraction corresponding to the TorD protein (Fig 3E) In addition, the TorDBlue preparation was subjected to MS analysis (Fig S2) Recombinant TorD was found to the dominant species in this preparation and was intact, except for partial modifications to the initiator methionine (Met-1), which are common in bacterial systems (Fig S2) Thus, from a total of 45 mg of recombinant TorDhis isolated by metal affinity chromatography and applied to the Cibacron Blue column, 2.3 mg of the TorDBlue protein harbouring all of the GTPase activity was recovered Further analysis of the two different TorD pools by SDS–PAGE and native PAGE suggested that the key difference lay in the oligomeric state of the proteins The TorD population that failed to bind to the CibacronTM Blue migrated at a low apparent molecular mass under native conditions (Fig 3B), displaying similar behaviour to the monomer form of TorD characterised by molecular exclusion chromatography (Fig 2) However, the GTPase-active TorDBlue clearly comprised oligomeric TorD (Fig 3B) The precise oligomeric state of the TorDBlue species was therefore investigated by analytical ultracentrifugation Sedimentation velocity analysis was performed on a sample taken from the initial nickel-affinity chromatography and this material was shown to contain predominantly monomeric TorD with detectable quantities of dimer and higher oligomeric species (Fig 4A) By contrast, however, analytical ultracentrifugation reveals that the GTPase-active TorDBlue species isolated here contains no monomeric TorD at all (Fig 4B) Instead, it was unequivocally established that TorDBlue is dominated by the homodimeric form of the protein (Fig 4B) Trace amounts of higherorder oligomeric species are also present in this sample (Fig 4B) Taken together, these data strongly indicate that gross overproduction of recombinant TorD results in a mixed population comprising numerous different oligomeric forms A small sub-population of proteins (approximately 5% by mass of the total) was found to adopt a stable homodimeric conformation that harbours a magnesium-dependent GTPase activity Kinetics of the GTPase reaction The Cibacron Blue affinity chromatography approach presented the opportunity to study the kinetics of GTP hydrolysis catalysed by the active TorD fraction For these experiments, Pi release from GTP hydrolysis by TorDBlue was assayed continuously using the EnzChekÒ Phosphate Assay Kit (Invitrogen, Carlsbad, 516 Fig The homodimer form of TorD dominates the GTPase-active fraction Sedimentation velocity analytical ultracentrifugation profiles of (A) the TorD pool immediately following metal affinity chromatography at 0.5 mgỈmL)1, rmsd = 0.024682, f ⁄ f0 = 1.199770, and (B) the TorDBlue pool immediately following Cibacron Blue affinity chromatography at 0.5 mgỈmL)1, rmsd = 0.024537, f ⁄ f0 = 1.201629 Samples at 0.25 and 0.75 mgỈmL)1 were also analysed (not shown) and gave similar profiles CA, USA) This coupled assay is based on the protocol described by Webb and Hunter [33] whereby, in the presence of Pi, 2-amino-6-mercapto-7-methylpurine riboside is converted by purine nucleoside phosphorylase to ribose-1-phosphate and 2-amino-6-mercapto7-methylpurine The product is detected by an increase in A360 The GTPase activity of TorDBlue in a range of GTP concentrations was assayed in 96-well plates, the Pi-dependent product measured continuously at A360 and a curve plotted of the initial velocities (V0) against the substrate concentration (Fig 5A) The best fit to FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS D Guymer et al A The E coli TorD homodimer has GTPase activity hydrolyse GTP with a Km of 1.42 mm and a Kcat of 3.9 min)1, both contributing to a very low specificity constant (Kcat ⁄ Km) of 45.77 M)1Ỉs)1 TorD residue D-68 confers substrate specificity B Fig Kinetics of GTP hydrolysis by the TorD homodimer (A) A linear plot of initial reaction rate, V0 (released Pi; lMỈmin)1) against GTP concentration (mM) plotted using ORIGIN software (OriginLab Corporation, Northampton, MA, USA) Aliquots (10 lM) of a pooled fraction of TorDBlue isolated by Cibacron Blue affinity chromotagraphy were asayed in a 96-well plate format at 37 °C with shaking in the presence of increasing amounts of substrate and the formed product measured continuously at A360 using the EnzCheck assay (B) A Hill plot of the same data shown in (A) converted into log (V0 – Vmax ⁄ V0) against log[GTP] Vmax was estimated as 39 lMỈmin)1 The straight line was calculated by Microsoft Excel (Microsoft Corp., Redmond, WA, USA) using liner regression: y = 2.1302x – 0.3237 (R2 = 0.9478) From these data, the Hill coefficient (equal to the gradient of the line), h = 2.13, Km [10(y ⁄ )h)] = 1.42 mM, Kcat = 3.9 min)1, and Kcat ⁄ Km = 45.77 M)1Ỉs)1 these data resulted in a sigmoidal curve (Fig 5A), indicative of a co-operative substrate binding and hydrolysis model with non Michaelis–Menten kinetics To extract the kinetic parameters from these data, a Hill plot (Fig 5B) was drawn of V0)(Vmax ⁄ V0) against log10[GTP], using an estimate for Vmax of 39 lmỈmin)1 obtained from the linear plot (Fig 5A) A line of best fit revealed the values for Km and the Hill coefficient, h A value of 2.13 for h was obtained, which indicates positive co-operativity of GTP binding and hydrolysis by the TorD homodimer TorD was calculated to Analysis of the primary amino acid sequence data suggests that TorD differs substantially from any canonical GTPase enzymes For example, regulatory GTP hydrolases (‘G-proteins’) all possess a conserved guanine nucleotide-binding domain, with the five polypeptide loops that comprise the guanine nucleotide-binding site being the most highly conserved elements that define the GTPase superfamily [31] These five loops are designated G-1 to G-5, with G-1, G-3 and G-4 being present in all canonical GTPases [34,35] G-1 corresponds to the ‘Walker A motif’ [36] and includes the consensus sequence Gx4GK[S ⁄ T] G-2 (DxnT) is a structurally mobile element and, in many GTPases, GTP-binding alters the conformation of this loop, bringing a conserved threonine essential for Mg2+ co-ordination into a position to facilitate GTP hydrolysis [35] G-3 corresponds to the ‘Walker B motif’ [36] and includes the consensus sequence Dx2G [35] Although the G-1 and G-3 consensus motifs are found in many nucleotide-binding proteins, it is the G-4 motif that provides the specificity for guanine nucleotides The characteristic sequence motif of G-4 is [N ⁄ T][K ⁄ Q]xD [34,37,38] and it is often preceded by a stretch of four hydrophobic or nonpolar amino acids [35] Finally, G-5 ([T ⁄ G][C ⁄ S]A) is less well conserved than the other motifs and cannot always be unambiguously identified from primary sequence data alone [35] TorD lacks each of the canonical G-1 and G-3 motifs that are considered essential for GTP recognition and hydrolysis However, examination of the amino acid sequence reveals a potential candidate for a G-4 guanine specificity motif in TorD Praefcke et al [39] described the human guanylate-binding protein that has guanine specificity conferred by a G-4 motif with the sequence TLRD In addition, the homologous GBP in chicken contained a TVRD motif at this position This is of particular interest because the E coli TorD possesses a TVRD tetrapeptide at positions 65– 68, which is predicted to be located on an exposed loop between helix and helix The residues of this putative G-4 motif were subjected to site-directed mutagenesis The focus was residue D-68, the final residue in the TVRD motif, because examples have been described where substitutions at this position have altered the substrate specificity of canonical GTPases [34,37,39–42] In classical GTPases, the aspartate is considered to form a hydro- FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS 517 The E coli TorD homodimer has GTPase activity D Guymer et al NTPase activity (% GTPase) 120% 100% 80% 60% 40% 20% 0% wtTorD – GTP wtTorD – XTP D68N – GTP D68N – XTP B 4.0 3.5 3.0 [Pi] mM 2.5 2.0 1.5 1.0 0.5 0.0 10 12 Elapsed time (h) wtTorD + GTP C wtTorD + GTP + XMP D68N + GTP D68N + GTP + XMP 120% 100% 80% 60% 40% 20% 0% P ITP ITP ATP ATP + GT GTP rD +wtTorD + wtTorD + D68W D68W + D68W + A TorD D68W variant is defective in the Tat proofreading process wtTo The physiological role of TorD residue D-68 was tested in vivo TorD has two physiological functions that can be independently measured and assays have been developed to study the overall biosynthesis of the TorA enzyme, as well as the isolated Tat proofreading activity First, the ability of the torD gene to rescue TMAO reductase activity in a DtorD mutant when expressed in trans was explored A chromosomal deletion strain (FTD100) was transformed with pUNIPROM derivatives encoding native TorD and TorDD68W The strains were grown anaerobically in LB supplemented with glycerol and TMAO and the benzyl viologen-linked TMAO reductase activity of periplasmic fractions was assayed (Fig 7A) TorDD68W was observed to support assembly of the periplasmic TMAO reductase activity to a level equivalent to native TorD (Fig 7A) 518 A NTPase activity (% GTPase) gen bond with the amino group at position of the guanine base, which is absent from the xanthine base [39,40] Thus, D-68 was substituted by asparagine and the hydrolytic activity of the TorDD68N variant towards GTP and XTP was measured using the malachite green assay (Fig 6A, B) TorDD68N was unaltered for GTPase activity, although this variant demonstrated enhanced XTPase activity (Fig 6A, B) To test for an increased affinity for xanthine nucleotides by a different means, the standard malachite green assay was modified to perform an assay measuring the effect of competition of a ten-fold excess of XMP on the GTPase activities of the native protein and the TorDD68N variant The GTPase activity of TorDD68N was found to be inhibited to a greater degree than the native TorD by the presence of excess XMP (Fig 6B) Examples exist of naturally-occurring GTPases where the G-4 consensus aspartate is substituted by tryptophan, and these are able to hydrolyse ITP in addition to GTP [34,37,41] A TorD D68W variant was therefore tested for its ITPase and ATPase activity (Fig 6C) The TorDD68W protein showed no obvious increase in ITPase activity compared to the native protein, which could clearly hydrolyse ITP already (Fig 6C) More interestingly, however, TorDD68W was observed to possess a hydrolytic activity towards ATP (Fig 6C), a substrate that native TorD was unable to recognise (Figs 1A and 6C) Taken together, these data implicate the TorD ‘G-4’ motif in playing a key role in substrate selectivity for this enzyme, especially with respect to the ability of the enzyme to distinguish between GTP and ATP Fig TorD residue D68 controls substrate specificity (A) The GTPase and XTPase activities of 0.1 mM ( 0.122 mg) native TorD and the D68N variant purified by immobilised metal affinity chromatography were assayed by the malachite green method in 50 lL reactions containing either mM GTP or XTP, 1.2 mM MgCl2, and 10 mM Tris–HCl (pH 7.5) Reactions were incubated at 22 °C for 24 h The results are shown as a percentage of the native TorD GTPase activity (B) A timecourse competition ⁄ inhibition assay of GTPase activity using 0.1 mM metal affinity chromatography-purified TorD and TorDD68N in 100 lL reactions containing 10 mM Tris–HCl (pH 7.5), 1.2 mM MgCl2 and mM GTP ± 50 mM XMP Phosphate release was quantified at h intervals by withdrawing 10 lL aliquots and subjecting those to the malachite green assay (C) Nucleotide hydrolysis activities of 0.1 mM samples of metal affinity chromatographypurified TorD and TorDD68W assayed by the malachite green method Each 50 lL reaction contained mM of either GTP, ATP, or ITP as well as 1.2 mM MgCl2 and 10 mM Tris–HCl (pH 7.5) Reactions were incubated at 22 °C for 24 h and the results are shown as a percentage of GTPase activity exhibited by native TorD In all cases, the error bars represent the SEM (n = 3) FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS D Guymer et al The E coli TorD homodimer has GTPase activity expression of active torD restores the Tat proofreading of this enzyme and so rescues hydrogenase-2 activity in the mutant strain RJ607 was transformed with a pUNIPROM vector encoding TorD and TorDD68W, grown anaerobically in LB supplemented with glycerol and fumarate, and benzyl viologen-linked hydrogenase-2 activity was assayed in whole cells (Fig 7B) Interestingly, TorDD68W was observed to have a reduced Tat proofreading activity in vivo Discussion Fig Residue D68 is involved in the Tat proofreading process Physiological activity of the TorDD68W variant (A) E coli strain FTD100 (DtorD) was transformed with a pUNIPROM vector expressing either torD (‘TorD’), or the D68W mutant (‘D68W’), and grown anaerobically in LB containing 0.5% (v ⁄ v) glycerol and 0.4% (w ⁄ v) TMAO before intact cells were assayed for TMAO:BV oxidoreductase activity (B) E coli strain RJ607 (/torA::hybO, DhybA) was transformed with a pUNIPROM vector expressing either torD (‘TorD’), or the D68W mutant (‘D68W’), and grown anaerobically in LB containing 0.5% (v ⁄ v) glycerol and 0.4% (w ⁄ v) fumarate before whole cells were assayed for hydrogen:BV oxidoreductase activity In all cases, the error bars represent the SEM (n = 3) Next, a specific assay for Tat proofreading was employed Jack et al [5] developed an assay based on a strain (RJ607) producing a TorA-signal-peptideHybO fusion protein Cells producing the TorA-HybO fusion have impaired hydrogenase-2 activity because assembly of the enzyme is disrupted; however, co- The crystal structure of Sh massilia TorD is a homodimer formed through 3D domain swapping [23] It has been established in the present study, in common with other TorD homologues [26,27], that E coli TorD can also be purified in a range of stable oligomeric forms, suggesting that oligomerisation may be a characteristic feature of the TorD family Most significantly, this work has now established the biochemical relevance of the dimerisation exhibited by TorD The E coli TorD homodimer displays an intrinsic specific GTPase activity, whereas the monomer form remains completely inactive In terms of kinetics, the sigmoidal curve of V0 versus [GTP] is indicative of a co-operative binding and hydrolysis model, and the Hill coefficient (h) of 2.13 implies a strongly positive co-operative binding model whereby binding and hydrolysis of GTP at one site enhances the affinity (or activity) towards GTP at other sites The Hill coefficient also enables an estimation of the number of hydrolytic sites present in the enzyme because current dogma states that h cannot be greater than the number of ligand-binding sites present within the molecule This suggests either that the active form of TorD could contain at least three active sites, or that the active oligomeric form could be larger than dimeric Indeed, the TorD protein from Sh massilia has been previously observed as stable trimers [27] Note that h provides only a minimum estimate for the number of binding sites Haemoglobin, for example, shows a Hill coefficient for oxygen binding varying in the range 1.7–3.0, but clearly possesses four binding sites [43,44] The discovery of an enzymatic activity associated with the domain swapped TorD dimmer, which is absent from the monomer, brings E coli TorD sharply into line with other biological systems that utilise domain swapping to regulate activity For example, glyoxylase I of Pseudumonas putida is a metastable domain-swapped dimer with two Zn2+ cofactors that also exists as a monomer with a significantly reduced activity and only a single Zn2+ cofactor [45] An additional example is the bleomycin resistance protein, FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS 519 The E coli TorD homodimer has GTPase activity D Guymer et al which sequesters two molecules of the bleomycin antibiotic within two crevices formed at the interface of the domain swapped dimer [46] Equally intriguing is the T7 endonuclease I, representing one of the most striking examples of a domain-swapped dimer, which comprises a composite catalytic site containing elements from both polypeptide chains [47,48] It is tempting to speculate that TorD dimerisation generates an analogous composite active site, which would enable the dimer to acquire a completely new biological function through domain swapping Indeed, such acquisition of a novel function would be consistent with the theory that domain swapping provides a model for the evolution of oligomeric enzymes [49,50] Although the mechanism of GTP hydrolysis by the TorD dimer remains to be elucidated, there are aspects of this activity that are mirrored in canonical nucleotide hydrolases For example, the GTPase activity of TorD is dependent upon the presence of magnesium ions and this is clearly in line with the requirements of the classical GTPases [29,30] Magnesium often functions as a phosphate ligand in canonical GTPases in addition to a variety of proposed activities involved in the actual hydrolysis reaction [30,31,51] Although the exact nature of the GTP-binding site remains to be determined, it is clear that TorD represents a novel family in this regard This is reinforced by the observation that active E coli TorD behaves very unusually on Cibacron Blue affinity media, eluting only under conditions of low ionic strength, perhaps suggesting hydrophobic interactions between the cofactor and protein dominate Interestingly, a more typical G-4 ‘guanine specificity’ loop was identified in TorD, and the D-68 side chain unequivocally was demonstrated to regulate substrate access to the active site(s) The E coli TorD sequence in question, 65-TVRD-68, is a close match to the G-4 consensus [N ⁄ T][K ⁄ Q]xD [34,37,38] This motif is conserved in the TorD and DmsD clades of the wider TorD family and is always located in an unstructured loop between helices and (Fig 8) The final aspartate in the TVRD tetrapeptide is occasionally naturally replaced by glutamine or glutamate (Fig 8G), although these side chains should still able to confer guanine specificity in classical GTPases [41] Note also that the TorD G-4 motif is often immediately followed by another conserved aspartate (Fig 8G), which could also have a role in determining nucleotide specificity This TorDD68W variant was unaffected in its GTPase activity but, in addition, showed a new ATPase activity The fact that this protein was defective in the Tat proofreading process is intriguing Why would ATP hydrolysis not be able to substitute for GTP hydrolysis in vivo? 520 Because the ATP hydrolysis reaction catalysed by the variant TorD form is obviously slower, and the concentration of ATP in the cell is higher than GTP, it is possible that ATP is inhibiting the Tat proofreading function of TorDD68W in vivo In an attempt to identify catalytic residues essential for GTP hydrolysis, extensive mutagenesis was conducted on E coli torD (Fig S3) based on recent biochemical and in silico experiments [5,19,20,24] However, from the 17 different TorD variants tested, none were found to be completely devoid of in vitro GTPase activity (Fig S3) TorD variants Q7L, as originally identified by Buchanan et al [19], and F41W [20] showed the lowest hydrolytic activity towards GTP (Fig S3), and these side-chains are in close proximity to the G-4 loop (Fig 8) What is the purpose of the GTPase activity of TorD? GTP hydrolysis is often used to regulate processes where multiple proteins must function co-ordinately [52] and the maturation of TorA is certainly a complex process involving the co-ordination of the two distinct functions of TorD with the folding of TorA and possible interactions of both proteins with the MGD biosynthetic apparatus [12] The concentration of GTP in the cytoplasm of an exponentially growing E coli cell has been estimated at approximately 0.9 mm [53] This is below the Km of GTP hydrolysis by TorD and perhaps suggests that TorD has only a low level of this activity in vivo Most GTPases bind very stably to GTP and have a very low intrinsic level of GTPase activity, and also require the action of GTPase activating proteins (GAPs), or the interaction with a specific effecter, to catalyse the hydrolysis of GTP The transition from GTP- to GDP-bound forms can therefore be limited by the intrinsic rate of GTP hydrolysis, by the action of a GAP, or by interaction with a cognate target GTPases governed by these factors are often referred to as ‘clocks’, ‘switches ⁄ adaptors’ and ‘sensors’, respectively [31] The turnover number of TorD for GTP of 3.9 min)1 is within the range expected for classical GTPases [31] but is very low in general terms, and it is possible that TorD may function as a ‘clock’ with the low rate of GTP hydrolysis coinciding with the maturation rate of TorA Another possibility is that arginines of the signal peptide, or perhaps residue R22 of TorD [20], may be contributing an ‘arginine finger’, thus activating the GTPase activity in response to signal binding Arginine fingers have been observed within the Ffh-FtsY composite active site and provided by the Ras and Rho GTPase GAPs [54–56] However, the experiments conducted in the present study were unable to demonstrate an increase in the rate of GTP hydrolysis in FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS D Guymer et al The E coli TorD homodimer has GTPase activity A B D C E F G Fig Putative GTP binding regions of TorD family proteins (A, B) Showing surface and cartoon representations, respectively, of the E coli TorD monomer model [19] The TVRD G-4 motif, T65-D68, is displayed in yellow together with other residues implicated in nucleotide binding (Q7, F41 and F180) [19,20,24] (C) The Sh massilia TorD homodimer [23] is shown as a surface representation with the two intertwined protomers coloured red and blue and the equivalent potential GTP-binding residues as highlighted in (A) and (B) (H8, W40, F182 and the G-4 loop K66-E69) displayed in yellow (D) A cartoon representation of a single functional unit of the Sh massilia TorD dimer (E, F) Showing cartoon and surface representations, respectively of S enterica serovar Typhimurium DmsD [24], again with predicted GTP-binding residues D8, L37, F175 and the G-4 loop T62-E66 displayed in yellow All images were generated using PYMOL (http://www.pymol.org) (G) Multiple sequence alignment of partial TorD sequences from different bacteria (from top to bottom: E coli, Shigella dysenteriae, S enterica serovar Typhimurium, Vibrio fischeri, Vibrio cholerae, Sh masillia, and Pastuerella multicoda) showing overall conservation of the G-4 loop tetrapeptide together with the adjacent C-terminal residue (boxed) Alignments were generated using CLUSTALW2 (http://www.ebi.ac.uk/tools/ clustalw2) and BOXSHADE (http://www.ch.embnet.org/software/BOX_form.html) response to addition of excess TorA signal peptide (data not shown) There is currently no evidence for the requirement of a specific GAP for TorD GTPase in vitro, although it remains a possibility that such may exist in vivo and further work to identify potential interacting partners will be required to investigate this The recent finding that TorD can bind the molybdenum cofactor, and may also interact with MobA in addition to two sites on apoTorA [12], suggests that TorD is intimately involved with all stages of TorA maturation The possibility that the GTP-binding pocket could also represent the binding site for the GTP-derived molybdenum cofactor should not be discounted; however, in line with other biological systems discussed above, it appears more likely that the magnesium-dependent GTP hydrolysis activity is regulating the intra- and inter-molecular interactions exhibited by the TorD protein FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS 521 The E coli TorD homodimer has GTPase activity D Guymer et al tion coefficients of 30 940 MỈcm)1 66 350 MỈcm)1 for FMalE-TorAhis Experimental procedures for TorDhis and Plasmids Overproduction of TorDhis was achieved from plasmid pQI-TorD [19], which incorporates a C-terminal hexahistidine tag onto the protein For constitutive expression of torD, plasmids pSU-TorD [5] and pUP-TorD [5], derivatives of pSUPROM and pUNIPROM [5], respectively, were used Point mutations were introduced into the torD sequence by site directed mutagenesis using the Quikchange protocol (Stratagene, La Jolla, CA, USA) Protein production and purification methods The host strain for TorDhis overproduction was E coli C43 (DE3) [57] harbouring pREP4 (lacI+, KanR; Roche Diagnostics, Basel, Switzerland) Protein production was performed in LB medium supplemented with appropriate antibiotics at 37 °C Cells were grown with shaking until D600 of  0.5 was reached followed by a further h under ‘inducing’ conditions in the presence of mm (final) isopropyl b-d-thiogalactoside Following overproduction, cells were harvested by centrifugation, resuspended in 20 mm Tris–HCl (pH 7.6) and mm dithithreitol, and broken by two passages through a French pressure cell at 8000 psi Debris and unbroken cells were removed by and centrifugation and the supernatant, which contains soluble proteins and membrane vesicles, was applied to a mL of a HisTrapÔ IMAC column (GE Healthcare, Milwaukee, WI, USA) equilibrated with 20 mm Tris–HCl (pH 7.6), 150 mm NaCl, 25 mm imidazole (Fluka, Buchs, Switzerland) and mm dithiothreitol Bound proteins were eluted using a 30 mL linear gradient in the range 20–500 mm imidazole in the same buffer Samples containing TorDhis as judged by SDS–PAGE were pooled and concentrated in a Vivaspin (Millipore, Billerica, MA, USA) device with a 10 kDa molecular mass cut-off Overproduction of /MalE-TorAhis [58] was performed in the same manner as for TorDhis, except the host strain was BL21 (DE3) [59] containing pREP4, and purification was performed as previously described [58] Molecular exclusion chromatography was performed using a SuperdexÔ 75 10 ⁄ 30 column (GE Healthcare) equilibrated in 20 mm Tris–HCl, 150 mm NaCl and mm dithiothreitol Cibacron blue affinity chromatography was performed using a mL HiTrapÔ Blue HP column (GE Healthcare) in a protocol adapted specifically for TorD in the present study The column was first equilibrated with 20 mm Tris– HCl (pH 7.5), 500 mm NaCl, mm dithiothreitol and IMAC-purified TorDhis was applied in this buffer Following re-equilibration, bound protein was eluted in a single step from loading buffer to deionised water Protein concentrations of purified preparations were determined by measuring A280 and applying molar extinc522 Protein analysis The SDS–PAGE method was as described by Lammli ă [60] and non-SDS-PAGE followed the same protocol but omitted SDS at every stage Western immunoblotting was performed as described previously [61] Analytical ultracentrifugation (sedimentation velocity) Proteins were prepared as 1.0 mgỈmL)1 stock solutions and diluted to give three samples at 0.25, 0.50 and 0.75 mgỈmL)1 in 50 mm Tris–HCl (pH 7.5) The samples were loaded into assembled cells with quartz windows and twosector 12 mm path length flow-through charcoal-filled epon centrepieces Each cell was manually fitted and aligned in an An50-Ti rotor (Beckman Coulter, Fullerton, CA, USA) The rotor was allowed to reach 16 °C and then equilibrated at that temperature for h before accelerating to 150 000 g Data were collected using the absorbance detection system of a XL-I analytical ultracentrifuge (Beckman Coulter) and the cells were scanned at 280 nm every 120 s Two hundred scans were collected for each concentration of protein The data were analysed using the sedfit [62,63] Solvent density and solvent viscosity were calculated using the public domain software sednterp (http://www.rasmb.bbri.org) The c(s) continuous distribution was generated in sedfit and then optimised using the meniscus and f ⁄ f0 as floating parameters The relative concentration and weight-average sedimentation coefficient were determined for each species using the integration tool in sedfit GTPase actvity assays Two GTPase assays were employed in the present study, a discontinuous assay based on detecting released phosphate using malachite green, and a continuous coupled reaction available commercially as a kit For the discontinuous assay, 0.1 mm samples of purified protein were incubated (typically overnight) in a reaction mixture containing 10 mm Tris–HCl (pH 7.5), mm GTP and 1.2 mm MgCl2 at 37 °C On completion, samples were diluted 250-fold in water and mixed with malachite green solution [0.7 m HCl, 0.3 mm malachite green oxalate, 8.3 mm Na2MoO4, 0.05% (v ⁄ v) Triton X-100] in a : ratio Following a 15 incubation at 22 °C, A630 was measured and compared with a phosphate standard curve prepared using identical solutions For the continuous assay, the activity of 10 lm TorDBlue was determined using the EnzChekÒ Phosphate Assay Kit FEBS Journal 277 (2010) 511–525 ª 2009 The Authors Journal compilation ª 2009 FEBS D Guymer et al (Invitrogen) Reactions were started by the addition of protein and phosphate release was measured continuously using a Synergy Plate reader (BioTek Inc., Winooski, VT, USA) set to measure A360 Absorbance data were converted into released Pi (lmỈmin)1) by comparison with a standard curve of known phosphate concentration All nucleotides were supplied by Sigma-Aldrich (St Louis, MO, USA), with the exception of XTP, which was supplied by Jena Bioscience GmbH (Jena, Germany) GTP, ITP, GMP and XMP were stored as 50 mm stocks in 10 mm Tris–HCl (pH 8.0) ATP was buffered in 75 mm Tris–HCl (pH 8.0) XTP was supplied as a 10 mm solution buffered to pH 7.5 In vivo Tat proofreading and TMAO reductase activity assays The signal peptide binding-dependent Tat proofreading activity of TorD can be isolated and assayed in vivo, as described previously [5] Strain RJ607, which carries a chromosomal torA::hybO fusion, was grown anaerobically in LB medium supplemented with 0.5% (v ⁄ v) glycerol and 0.4% (w ⁄ v) fumarate and washed whole cells assayed for hydrogen:benzyl viologen oxidoreductase activity as described previously [5] Periplasmic TMAO reducatase activity was assayed in strain FTD100 (DtorD) [5] The strain was grown anaerobically in LB medium supplemented with 0.5% (v ⁄ v) glycerol and 0.4% (w ⁄ v) TMAO, harvested by centrifugation, and separated into periplasmic fraction and sphaeroplasts using a lysozyme ⁄ EDTA method [64] TMAO-dependent oxidation of reduced benzyl viologen was then assayed as described previously [65] Acknowledgements We thank Tracy Palmer (Dundee) for useful discussions and Martin Zoltner (Dundee) for help with analysing the kinetic data Mass spectrometry was carried out by Kenneth Beattie and Samantha Kosto of the Fingerprints Proteomics Facility at the University of Dundee This work was funded in the UK by the BBSRC through a Doctoral Training Grant awarded to the University of Dundee, and J.M was supported by the Swiss National Science Foundation References Berks BC, Palmer T & Sargent F (2003) The Tat protein translocation pathway and its role in microbial physiology Adv Microb Physiol 47, 187–254 Berks BC (1996) A common export pathway for proteins binding complex redox cofactors? 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Tat proofreading and TMAO reductase activity assays The signal peptide binding-dependent Tat proofreading activity of TorD can be isolated and assayed in vivo, as described previously [5] Strain... protein peak isolated by the ‘reverse’ Blue Sepharose chromatography protocol was analysed Fig TorD GTPase activity can be isolated by Cibacron Blue affinity chromatography (A) Unusual behaviour of TorD