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Originally published In Press as doi:10.1074/jbc.M703464200 on June 27, 2007

J. Biol. Chem., Vol. 282, Issue 35, 25569-25576, August 31, 2007
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The ATPase Activity of the DNA Transporter TrwB Is Modulated by Protein TrwA

IMPLICATIONS FOR A COMMON ASSEMBLY MECHANISM OF DNA TRANSLOCATING MOTORS*

Irantzu Tato1, Inmaculada Matilla, Ignacio Arechaga, Sandra Zunzunegui, Fernando de la Cruz2, and Elena Cabezon3

From the Departamento de Biología Molecular, Universidad de Cantabria (UC) e Instituto de Biomedicina y Biotecnología de Cantabria, IBBTEC (CSIC-UC-IDICAN), 39011 Santander, Spain

Received for publication, April 25, 2007 , and in revised form, June 20, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Conjugative systems contain an essential integral membrane protein involved in DNA transport called the Type IV coupling protein (T4CP). The T4CP of conjugative plasmid R388 is TrwB, a DNA-dependent ATPase. Biochemical and structural data suggest that TrwB uses energy released from ATP hydrolysis to pump DNA through its central channel by a mechanism similar to that used by F1-ATPase or ring helicases. For DNA transport, TrwB couples the relaxosome (a DNA-protein complex) to the secretion channel. In this work we show that TrwA, a tetrameric oriT DNA-binding protein and a component of the R388 relaxosome, stimulates TrwB{Delta}N70 ATPase activity, revealing a specific interaction between the two proteins. This interaction occurs via the TrwA C-terminal domain. A 68-kDa complex between TrwB{Delta}N70 and TrwA C-terminal domain was observed by gel filtration chromatography, consistent with a 1:1 stoichiometry. Additionally, electron microscopy revealed the formation of oligomeric TrwB complexes in the presence, but not in the absence, of TrwA protein. TrwB{Delta}N70 ATPase activity in the presence of TrwA was further enhanced by DNA. Interestingly, maximal ATPase rates were achieved with TrwA and different types of dsDNA substrates. This is consistent with a role of TrwA in facilitating the interaction between TrwB and DNA. Our findings provide a new insight into the mechanism by which TrwB recruits the relaxosome for DNA transport. The process resembles the mechanism used by other DNA-dependent molecular motors, such as the RuvA/RuvB system, to be targeted to the DNA followed by hexamer assembly.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Conjugative DNA transfer is an efficient way for bacteria to acquire new genetic information. Any DNA molecule containing a short segment called origin of transfer (oriT)4 can be conjugatively transferred to a recipient cell if a conjugation machinery is provided. This machinery consists of two functional subsets of proteins: Dtr proteins (involved in the processing of conjugative DNA) and Mpf proteins (involved in the production of exocellular pili and the formation of a channel structure for secretion). Both protein subsets are encoded by the transfer region of a conjugative plasmid (1).

R388 plasmid displays one of the simplest transfer regions in Gram-negative bacteria, coding for just three Dtr proteins: TrwA, TrwB, and TrwC. TrwC is a two domain (relaxase and DNA helicase) protein. Its relaxase domain introduces a site-specific nick at oriT and remains covalently bound to the 5'-end of the DNA strand that is transferred. Therefore, TrwC acts as a pilot protein in the transfer of the resulting nucleoprotein complex. Recent work demonstrated that TrwC still plays an essential role in the recipient cell, where it performs the strand-transfer reaction required to recircularize the transferred DNA and thus, terminates conjugation (2, 3). The atomic structure of the relaxase domain of TrwC was solved (4), showing a fold shared by the relaxase of plasmid F (5) and replication initiation proteins of some viruses (6, 7). TrwA is a tetrameric oriT-specific DNA-binding protein that belongs to the ribbon-helix-helix protein family DNA-binding proteins (8). Experimental data revealed that TrwA binds to two specific sites in R388 oriT: sbaA and sbaB. Binding to these sites enhances TrwC relaxase activity and represses trwABC operon transcription (9). TrwA consists of two structural domains: an N-terminal domain, involved in DNA binding and a C-terminal domain, responsible for TrwA tetramerization (8). Recently, the structure of the C-terminal domain of the related protein TraM from plasmid F was solved (10). The nucleoprotein complex formed by oriT, relaxase TrwC, host protein IHF, and protein TrwA is called the relaxosome, which must be recruited to the secretion channel for DNA transfer. The third Dtr protein, TrwB, is an inner membrane protein that couples the relaxosome to a type IV secretion system (T4SS), a multiprotein complex that spans the inner and outer membranes. In R388 plasmid, the T4SS consists of eleven proteins (TrwD to TrwN). Direct interactions between TrwB and TrwE, an inner membrane protein, were described (11) and corroborated with protein homologues in IncH plasmids (12). On the cytoplasmic side, the coupling protein interacts with relaxosomal components. In R388, interactions between TrwB and the relaxosome occur via TrwA and TrwC (11). In IncF plasmids, interactions between the coupling protein TraD and TraM have also been reported (13, 14). Besides its coupling role in bacterial conjugation, TrwB is also thought to work actively as a DNA transporter. The crystallographic structure of TrwB{Delta}N70, the cytoplasmic domain of TrwB, revealed a hexamer with 6-fold symmetry and a central channel of about 20 Å in diameter (15). The striking structural similarity between TrwB and other well known molecular motors, such as F1-ATPase (16) or T7 gene 4 helicase (17), suggested that TrwB might also operate as a motor, pumping DNA through its central channel using energy derived from ATP hydrolysis. This hypothesis is strengthened by recent biochemical data, which demonstrate that TrwB is a DNA-dependent ATPase (18). The protein assembles as an oligomer on ssDNA and exhibits positive cooperativity for ATP hydrolysis. Following this idea, a mechanism similar to the so-called "binding change mechanism" assigned to F1-ATPase, was proposed for TrwB (19).

In this work, we have extended the analysis of TrwB ATPase activity by using different DNA substrates. We have also observed that TrwB ATPase activity is stimulated by protein TrwA, revealing specific interactions between the two proteins. This modulation might facilitate the dynamic coupling between TrwB and DNA during the conjugative process. These findings shed light on the coupling role assigned to TrwB (20) and on the mechanism by which this protein recruits the relaxosome for conjugative transport. The system resembles the mechanism used by other DNA-dependent molecular motors, such as the RuvA/RuvB recombination system (21), to be targeted and assembled as hexamers on the DNA, leading us to propose a common mechanism of assembly for hexameric molecular motors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Purification and Preparation of DNA Substrates—Purification of protein TrwB{Delta}N70 was carried out as described previously (18). TrwA-derived proteins containing a C-terminal His tag were purified as follows. Strains used were derivatives of Escherichia coli C41 (22) containing plasmids pSU1547, pSU1548, and pSU1550, which overexpress TrwA-His6, TrwA-(2–73)-His6 and TrwA-(36–121)-His6, respectively (8). Bacteria were grown in 2 liters of LB medium. After addition of isopropyl-1-thio-beta-D-galactopyranoside (Apollo), cells were incubated for 8 h at 37°C, harvested, and resuspended in 40 ml of buffer A (100 mM Tris-HCl, pH 7, 500 mM NaCl, 0.001% PMSF) supplemented with 2.5 mM benzamidine. Cells were lysed by sonication (Vibra Cell, Sonic & Materials Inc.), and the lysate was centrifuged at 138,000 x g for 30 min at 4 °C. The supernatant was then loaded to a His trap HP (Amersham Biosciences) chromatographic column, equilibrated with buffer A. Bound proteins were eluted in a 50-ml imidazole gradient (0–500 mM) with buffer B (100 mM Tris-HCl, pH 7, 200 mM NaCl, 500 mM imidazole, 0.001% PMSF). TrwA-containing fractions were pooled and concentrated in a Centricon YM10 (Amicon) concentrator to 4 ml. Then, protein was loaded on a HiLoad 16/60 Superdex 75 column (Amersham Biosciences) equilibrated with buffer 50 mM Pipes-NaOH, pH 6.2, 100 mM NaCl, 2 mM MgCl2, 0.1 mM EDTA, 10% glycerol and 0.001% PMSF. Protein was stored at 4 °C. Protein concentrations were determined by the method of Bradford (Bio-Rad).

Circular DNA substrates, obtained by using Qiagen plasmid DNA purification kit, were vector plasmid pUC8 (23) and pSU1186 (24), which contains R388 oriT cloned in pUC8. Additionally, a linear DNA fragment corresponding to nucleotides 15934–16622 from the R388 oriT DNA sequence (GenBankTM accession no BR000038 [GenBank] ) was obtained by PCR amplification with appropriate oligonucleotides. Circular ssM13DNA was prepared as described in Ref. 25. The 45-mer oligonucleotide used (5'-TCG CCA CGT TTC GCC GTT TGC GGG GGT TTC TGC GAG GAA CTT TGG-3') was described previously (18). ssDNA and dsDNA concentrations of the stock solutions were measured by absorbance at 260 nm, using 40 µg·ml-1·A260-1 and 50 µg·ml-1·A260-1, respectively, as conversion factors.

Nucleotide Hydrolysis Assays—ATP hydrolysis was analyzed by a coupled enzyme assay as described previously (18). To analyze the effect of increasing concentrations of ssDNA and dsDNA or the effect of TrwA-derived proteins on TrwB{Delta}N70 ATPase activity, TrwB{Delta}N70 was preincubated with the different substrates for 10 min at 37 °C in 150 µl of ATPase assay mixture, consisting of 50 mM Pipes-NaOH, pH 6.2, 75 mM NaCl, 6 mM MgCl, 10% glycerol, 0.5 mM phosphoenolpyruvate, 0.25 mM NADH, 60 µg/ml pyruvate kinase, and 60 µg/ml lactate dehydrogenase (Roche Applied Science). Reactions were started by addition of ATP, unless otherwise indicated.

Gel Filtration Analysis—Protein samples were chromatographed at 20 °C at a flow rate of 30 µl/min on a Superdex-200 PC 3.2/30 column (Amersham Biosciences SMART system). The column was equilibrated in buffer containing 50 mM Pipes-NaOH pH 6.2, 100 mM NaCl, 2 mM MgCl2, 0.1 mM EDTA, 10% glycerol, and 0.001% PMSF. Absorbance was monitored at 280 nm. Samples (40 µl) of TrwB{Delta}N70 (208 µg), TrwA-(36–121)-His6 (343 µg), and the mixture of both proteins were analyzed. A2-µl sample from each fraction was run on a 15% SDS-polyacrylamide gel, and proteins were detected by Coomassie Blue staining.

Electron Microscopy and Image Analysis—TrwB{Delta}N70 (60 µM) was preincubated with the 45-mer oligonucleotide and chromatographed on a Superdex-200 PC 3.2/30 at 20 °C, as described previously (18). A 5-µl sample from a fast-migrating TrwB{Delta}N70-DNA complex, with an estimated molecular mass of 440 kDa, was taken for electron microscopy. For the analysis of TrwB-TrwA complexes, TrwB{Delta}N70 (208 µg) and TrwA-(36–121)-His6 (343 µg) proteins were incubated for 10 min at room temperature in buffer 50 mM Pipes-NaOH pH 6.2, 100 mM NaCl, 2 mM MgCl2, 0.1 mM EDTA, and 10% glycerol. Then, the sample was diluted in buffer to a protein concentration of 0.01 mg/ml and, immediately, samples were taken for the analysis.

Aliquots (5 µl) were applied to glow-discharged carbon-coated grids and stained for 1 min with 2% uranyl acetate. Images were recorded on Kodak SO163 films at x60,000 nominal magnification in a JEOL 1200EX-II electron microscope operated at 100 KV. Micrographs were digitized in a Zeiss SCAI scanner with a final sampling rate of 2.33 Å/pixel. A total of 2,463 TrwB-DNA particles (or 776 particles in the case of TrwB-TrwA complexes) were selected and normalized using XMIPP image processing software (26). Alignment and classification was performed by maximum likelihood multi-reference refinement methods (27). Kernel probability density estimator self-organizing maps (28) were used for further classification of particles. Final classes consisted of averages of 150–200 particles.


Figure 1
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  		FIGURE 1.
Effect of ssDNA and dsDNA on TrwB{Delta}N70 ATPase activity. Reactions contained TrwB{Delta}N70 (3 µM as monomer), 5 mM ATP, and increasing concentrations of ssDNA or dsDNA substrates, represented as micromolar concentrations of bases (for ssDNA) or base pairs (for dsDNA). (•) ssM13 DNA, ({diamondsuit}) PCR fragment of 688 bp, ({blacktriangleup}) pUC8 vector, ({blacksquare}) plasmid pSU1186. Reactions were carried out with the coupled spectrophotometric assay. Each figure represents the average of three different experiments.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TrwB{Delta}N70 ATPase Activity Is Stimulated by Both ssDNA and dsDNA—In an earlier work, we characterized a DNA-dependent ATPase activity of TrwB{Delta}N70 on ssDNA substrates, such as circular M13 ssDNA or oligonucleotides (18). In this study, we have further characterized TrwB ATPase activity by using dsDNA substrates with different DNA topologies (circular supercoiled and relaxed forms as well as linear dsDNA). Results show that ssDNA and dsDNA both stimulate TrwB{Delta}N70 ATP hydrolysis (Fig. 1). Both types of substrates lead to similar Vmax values (~500 nmol min-1 mg-1). When acting on ssDNA substrates, TrwB ATPase activity showed a dependence on the absolute DNA concentration (hydrolysis rates reached a maximum and, at higher ssDNA concentrations, started to decline) (18). Fig. 1 compares this result with those obtained when using dsDNA with different topologies. Interestingly, the inhibitory effect at high DNA concentration was not observed with dsDNA substrates.

Some sequence-dependent DNA translocases, such as FtsK, move along DNA responding to directing DNA sequences (29). Additionally, some hexameric replicative helicases, such as E1 from papillomavirus, also show specificity for DNA binding at the viral origin (30). Therefore, we decided to check for DNA sequence dependence in TrwB ATPase activity. Because conjugative DNA processing starts at a specific sequence (oriT), we used different dsDNA substrates containing or lacking the oriT sequence: cloning vector pUC8, plasmid pSU1186 (R388 oriT cloned into pUC8) and M13 dsDNA. No difference in the kinetics of ATP hydrolysis was observed between the three dsDNA substrates (Fig. 1). Closed circular supercoiled and relaxed DNA substrates were used to check a putative dependence on DNA topology. Both types of substrates were as efficient as the 688-bp linear dsDNA fragment obtained by PCR (data not shown).

TrwA Stimulates the ATPase Activity of TrwB{Delta}N70—TrwB has a role in recognizing the conjugative relaxosome through specific protein-protein interactions (20). The protein is anchored in the inner membrane; therefore, specific binding of TrwB to the relaxosome can position the nucleoprotein complex at the membrane interface of the transport machinery. We therefore examined whether interactions with the conjugative proteins forming the relaxosome of R388 plasmid TrwA and TrwC had any effect on TrwB ATPase activity.

TrwA-His6 alone did not show ATPase activity either in the presence or absence of DNA (data not shown). TrwB{Delta}N70 protein alone had a weak ATPase activity of about 30 nmol min-1 mg-1, as described previously (18), which increased to 550 nmol min-1 mg-1 upon the addition of ssDNA. We first examined whether TrwA-His6 had any effect on the ATPase activity of TrwB{Delta}N70. The effect of varying the concentration of TrwA-His6 on the rates of ATP hydrolysis in the absence of DNA and at a fixed concentration of TrwB{Delta}N70 is shown in Fig. 2A. At a fixed concentration of TrwB{Delta}N70 (0.3 µM monomer), ATP hydrolysis rates raise with increasing concentrations of TrwA, reaching a maximum of 1,300 nmol min-1 mg-1 at 0.2 µM TrwA-His6 tetrameric protein. Above this protein concentration, TrwB{Delta}N70 ATPase activity did not increase further. Similar experiments were carried out with TrwC. For these experiments we used the relaxase domain of TrwC, (TrwC-N293), which does not present ATPase activity. TrwC-N293 did not stimulate TrwB{Delta}N70 ATPase activity under any of the conditions tested (data not shown).

Fig. 2B shows the time course of TrwB{Delta}N70 mediated ATP hydrolysis in the presence of 0.2 µM TrwA-His6 tetrameric protein. Reactions were initiated either by the addition of TrwB{Delta}N70 protein or ATP. In the latter case, TrwB{Delta}N70 and TrwA-His6 proteins were incubated in the reaction buffer for 10 min, prior to the addition of ATP. Results show that steadystate rates of ATP hydrolysis are comparable in both cases. However, there was an evident lag phase when both proteins were not preincubated together. In that case, the time course was nonlinear for the first 10 min, suggesting that direct TrwA-TrwB protein interactions are required to observe linear progress of ATP hydrolysis.

TrwA Is More Effective in Enhancing TrwB{Delta}N70 ATPase Activity in the Presence of DNA—A kinetic analysis of TrwB{Delta}N70-mediated ATP hydrolysis in the presence of TrwA-His6 protein was carried out with different DNA substrates, with results summarized in Fig. 3. Addition of both TrwA-His6 and DNA greatly enhanced TrwB{Delta}N70 ATPase activity. Different DNA substrates were tested for their efficiency as cofactors in the enhancement of the TrwB{Delta}N70 ATPase activity by TrwA-His6. In all cases, ATP hydrolysis values were stimulated to a much higher extent than in the absence of TrwA-His6 protein (see Fig. 1 for comparison). At a fixed concentration of TrwB{Delta}N70 (0.3 µM TrwB{Delta}N70 monomer), the ATPase activity was enhanced about 10-fold by linear or supercoiled dsDNA and 5–6-fold by M13 ssDNA, in the presence of saturating amounts of TrwA-His6. Interestingly, the short 45-mer ssDNA oligonucleotide hardly increased ATPase rates above values obtained with TrwA-His6 in the absence of DNA (Fig. 3, panel B).


Figure 2
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  		FIGURE 2.
Effect of TrwA-His6 protein on TrwB{Delta}N70-mediated ATP hydrolysis in the absence of DNA. A shows the dependence of TrwB{Delta}N70 ATP hydrolysis on TrwA-His6 concentration. Reactions contained TrwB{Delta}N70 (0.3 µM as monomer) and increasing concentrations of TrwA-His6. Reactions were carried out with the coupled spectrophotometric assay, starting the reaction by the addition of 5 mM ATP, final concentration. B shows the time course of TrwB{Delta}N70 protein-mediated ATP hydrolysis in the presence of 0.2 µM tetrameric TrwA-His6. Reactions were carried out either by preincubation of the two proteins for 10 min, initiating the reaction by the addition of ATP (•) or without preincubation, initiating the reaction by the addition of TrwB{Delta}N70 protein ({blacktriangleup}).

 
The results obtained with relaxed and linear dsDNA were comparable to those obtained with supercoiled DNA. Moreover, there was no dependence on the sequence or length of the plasmid used. TrwA-His6 was able to stimulate TrwB{Delta}N70 ATPase activity independently of the presence or absence of its binding site on the oriT sequence (plasmid pSU1186 versus pUC8). The optimal TrwA/TrwB ratio was similar to that obtained in the absence of DNA.

The C-terminal Region of TrwA, but Not the N terminus, Stimulates TrwB{Delta}N70 ATPase Activity—To obtain clearer insights into the functional aspect of the domain organization of TrwA and how different domains can affect TrwB{Delta}N70 ATPase activity, we used truncated segments of TrwA containing the N-terminal region (residues 2–73) or the C-terminal region (residues 36–121). The ability of the truncated forms of the protein to bind oriT was previously analyzed, showing that TrwA-(2–73)-His6 forms a stable DNA-binding domain, which binds specifically R388 oriT, whereas TrwA-(36–121)-His6 does not bind DNA (8). The effect of each domain on TrwB{Delta}N70 ATPase activity was analyzed by using the same assay as used with the full-length protein TrwA-His6 (Fig. 4). Results show that TrwA-(2–73)-His6 had no effect on TrwB{Delta}N70 ATPase rates, neither in the absence nor in the presence of DNA (Fig. 4, A and B, respectively). On the other hand, hydrolysis of ATP was stimulated by TrwA-(36–121)-His6, but a higher ratio TrwA/TrwB was required to obtain the same stimulatory values observed with the full-length protein. Therefore, we conclude that TrwA residues within the region 73–121 are involved in interactions with TrwB, being these interactions responsible for modulating TrwB ATPase activity.

Gel Filtration Chromatography Reveals a Stable Protein Complex between TrwB and the C-terminal Domain of TrwA—The ability of TrwA to interact with TrwB was further analyzed by gel filtration chromatography (Fig. 5A). TrwB{Delta}N70 was incubated with TrwA-(36–121)-His6, as described under "Materials and Methods." After 10 min at room temperature, samples were loaded into the column. Gel filtration chromatography showed the interaction between the two proteins, because they eluted in a single peak with an estimated mass of 68 kDa (peak 3 in Fig. 5A). TrwA-(36–121)-His6 protein eluted as a tetramer, with an estimated mass of 56 kDa (peak 2) and TrwB{Delta}N70 eluted as a monomer, with an estimated mass of 51 kDa (peak 1). This result indicates that the complex between TrwB and the C-terminal domain of TrwA elutes with a 1:1 stoichiometry. The formation of the complex was confirmed by SDS-PAGE analysis of the resolved column fractions (Fig. 5B) and by cross-linking experiments (data not shown).


Figure 3
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  		FIGURE 3.
Effect of TrwA-His6 protein on TrwB{Delta}N70-mediated ATP hydrolysis in the presence of different DNA substrates. A, all reactions contained TrwB{Delta}N70 (0.3 µM as monomer), 5 mM ATP, 52 µM base ssDNA, or 43 µM bp dsDNA and increasing concentrations of TrwA-His6. Experiments were carried out with the coupled spectrophotometric assay, initiating the reaction by the addition of ATP. The effects of ssDNA ({diamondsuit}), supercoiled dsDNA ({blacksquare}), or linear dsDNA ({blacktriangleup}) are shown. For comparison, activity in the absence of DNA has also been represented (•). B represents the TrwB{Delta}N70 ATPase rates in the absence of TrwA-His6 protein (in black color) or at saturating TrwA-His6 protein concentrations (in gray color). Data are derived by averaging at least four experiments.

 


Figure 4
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  		FIGURE 4.
Effect of the N-terminal and C-terminal domains of TrwA-His6 on TrwB{Delta}N70 ATPase rates. Effect of two truncated domains of TrwA containing the N-terminal region (residues 2–73) or the C-terminal region (residues 36–121) on TrwB{Delta}N70-mediated ATP hydrolysis in the absence (A) or presence of 43 µM bp DNA from plasmid pUC8 (B). Experiments were carried out with the coupled spectrophotometric assay. Reactions contained TrwB{Delta}N70 (0.3 µM as monomer), 5 mM ATP and increasing concentrations of TrwA-His6 (•), TrwA-(2–73)-His6 ({blacktriangleup}), or TrwA-(36–121)-His6 ({blacksquare}).

 
DNA and TrwA Promote TrwB{Delta}N70 Oligomerization—Electron microscopic analysis of the samples (Fig. 6) revealed the presence of oligomeric TrwB{Delta}N70 complexes when the protein was first incubated with either TrwA or the 45-mer oligonucleotide (oligonucleotide shown previously to promote the oligomerization and ATPase activity of TrwB{Delta}N70) (18). The starting material for image analysis of TrwB-DNA complexes is shown in Fig. 6A. It corresponds to TrwB{Delta}N70-DNA complexes obtained by gel filtration, in a peak that elutes with an estimated molecular of 440 kDa. The oligomeric TrwB{Delta}N70 complex contains DNA, as shown by a correlated increase in the absorbance at 260 nm (18). After the alignment and classification of 2,463 of these particles, three class averages are shown in Fig. 6B. The size of these particles is approximately ~100 Å, which perfectly correlates to the size of hexameric TrwB{Delta}N70 complexes solved by crystallography (15). The averaged electron microscopic images shown in Fig. 6C correspond to oligomeric TrwB{Delta}N70 complexes obtained after incubating TrwB{Delta}N70 and TrwA-His6 proteins. In this case, samples were not previously chromatographed, since the formation of complexes in the presence of TrwA-His6 is dependent on protein concentration. The size of these particles is similar to that of the TrwB-DNA complexes. In the absence of DNA or TrwA, TrwB{Delta}N70 is in a monomeric state and no oligomeric assemblies could be visualized by electron microscopy (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TrwB is a DNA transporter that also acts as a coupling protein during bacterial conjugation, recruiting the relaxosome to the inner membrane, where the protein is anchored. Previous biochemical and structural analysis revealed a DNA-dependent ATPase activity for TrwB{Delta}N70 (18). However, the coupling mechanism between TrwB and the relaxosome remained unclear. Preliminary evidence for an interaction between TrwB and TrwA proteins was observed in co-elution experiments when TrwB was immobilized on an affinity column (11). The work presented here demonstrates that TrwA protein greatly stimulates the ATPase activity of TrwB{Delta}N70. Specific interactions between both proteins allow the modulation of TrwB{Delta}N70 ATPase activity by TrwA. As shown earlier (18), TrwB is an active ATPase in an oligomeric state, likely as a hexamer, and oligomerization is promoted by DNA binding. The results presented here suggest that TrwA also stimulates TrwB{Delta}N70 ATPase activity by promoting TrwB oligomerization, since time course experiments show a lag phase that disappears when both proteins are preincubated together. This idea is also supported by electron microscopy analysis, which shows oligomeric TrwB{Delta}N70 complexes when the protein is previously incubated with TrwA. At present, it is not possible to conclude if TrwA brings about the oligomerization of TrwB and then dissociates from the complex or whether it remains bound to the oligomer.

The major effect of TrwA protein was the enhancement of the effects of DNA on TrwB ATPase activity. Strikingly, in the presence of the TrwA protein, maximum values of ATPase activity were achieved with dsDNA substrates, which stimulated ATPase rates 10-fold. M13 ssDNA increased the activity by 5–6-fold, but the presence or absence of TrwA did not alter values exhibited with oligonucleotides (45- or 66-mer) (Fig. 3B). These results suggest that TrwB{Delta}N70 works on DNA with high processivity only in the presence of TrwA.

The different results obtained with ssDNA and dsDNA led us to analyze TrwB{Delta}N70 ATPase activity in the presence of different types of dsDNA substrates, and in the absence of TrwA. Another conclusion of this work is that TrwB{Delta}N70 ATPase activity is stimulated by both ssDNA and dsDNA. As it happened in the presence of TrwA, both linear and circular supercoiled dsDNA stimulated TrwB{Delta}N70 ATPase activity. Although the ATPase rates in the presence of dsDNA are high in vitro, the in vivo role for TrwB acting on dsDNA remains unclear, since it is generally accepted that ssDNA is the translocated substrate during conjugation (31, 32). This capability to work with both ssDNA and dsDNA has also been described for several hexameric helicases, such as DnaB or T7 phage gp4 helicase. These proteins unwind the DNA replication fork while encircling ssDNA, but are also able to actively translocate along a duplex, encircling both DNA strands (33). Other molecular motors, such as RuvB, involved in branch migration in the late stages of recombination, also share this capability (34). Therefore, this tracking capability on dsDNA might generalize to other DNA translocating motors, such as TrwB. Interestingly, both RuvB and T7 gp4 helicase work on DNA with a 5' to 3' polarity. Although in the case of TrwB, no helicase activity has been proven so far, DNA is also transferred in a 5' to 3' direction during bacterial conjugation. As discussed by Egelman (35), some proteins initially described as helicases, may use the energy of ATP hydrolysis to translocate along DNA or pump DNA without acting as classical unwinding enzymes.


Figure 5
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  		FIGURE 5.
Interactions between TrwB{Delta}N70 and TrwA-(36–121)-His6 determined by gel filtration chromatography. A, protein samples were chromatographed separately, as described under "Materials and Methods." Ve represents the elution volume. Run 3 was obtained after incubation for 10 min of proteins TrwB{Delta}N70 (208 µg) and TrwA-(36–121)-His6 (343 µg). A single peak (peak 3) was obtained, with an estimated mass of 68 kDa. Peaks 1 and 2 represent the separate elution profiles of TrwB{Delta}N70 (208 µg) and TrwA-(36–121)-His6 (343 µg), respectively. TrwA-(36–121)-His6 elutes as a tetramer, with an estimated mass of 56 kDa. TrwB{Delta}N70 elutes as a monomer, with an estimated mass of 51 kDa. B, SDS-PAGE of peak fractions. The first two lanes correspond to control lanes of TrwA-(36–121)-His6 (900 ng) and TrwB{Delta}N70 (600 ng). The third lane corresponds to molecular markers of 106, 77, 50.8, 35.6, 28.1, and 20.9 kDa. A 2.0-µl volume sample was taken from fractions 31–42 in each run. The gel on the top shows the fractions obtained from run 3. The gel at the bottom was obtained when 2.0-µl samples of equivalent fractions of runs 1 and 2 were mixed and then run together on a 15% SDS-polyacrylamide gel. Proteins were detected by Coomassie Blue staining.

 


Figure 6
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  		FIGURE 6.
Electron microscopy of TrwB{Delta}N70-DNA and TrwB{Delta}N70-TrwA complexes. A, original images of TrwB{Delta}N70-DNA complexes stained with uranyl acetate (scale bar, 100 nm). B and C, classes of averaged images (150–200 particles each) corresponding to TrwB{Delta}N70-DNA and TrwB{Delta}N70-TrwA complexes, respectively (scale bars, 5 nm).

 
How does TrwA modulate TrwB function as an ATP-dependent motor? Interactions between the two proteins occur via the C-terminal domain of TrwA, whereas its N-terminal domain has no effect on TrwB{Delta}N70 ATPase activity. Therefore, the interacting region is located in TrwA between residues 73–121. This result is in agreement with experiments carried out with the F-homologue protein TraM. They define the C-terminal domain of the protein as the domain involved in interacting with the coupling protein (13).

To observe the interaction between the C-terminal domain of TrwA and TrwB directly, we analyzed the complex by gel filtration chromatography. The 1:1 stoichiometry of the complex reveals that the mixture of the two proteins causes the dissociation of TrwA-(36–121) tetramers to produce a monomeric state complexed with TrwB. The formation of the complex is dependent on protein concentration and, at present, we cannot conclude whether the 1:1 stoichiometry corresponds to a pre-loading complex or arises from dissociation of a complex of higher oligomeric state. The latter possibility is supported by the electron microscopic images obtained with the starting material, which show the existence of oligomeric TrwB{Delta}N70 complexes.

Interactions between TrwA/TrwB proteins and the ability of TrwA to modulate TrwB{Delta}N70 ATPase activity is similar to the mechanism used by other DNA-dependent molecular motors, such as the RuvA/RuvB system, to be targeted and assembled as hexamers on the DNA. RuvA is a tetrameric protein that binds DNA at the Holliday junction and facilitates the loading of RuvB protein onto DNA. RuvA enhances the ATPase activity of RuvB, a hexameric molecular motor required for branch migration during homologous recombination. The C-terminal domain of RuvA (domain III) interacts with RuvB and modulates its ATPase activity. Furthermore, although the RuvA/RuvB motor works as a tetramer/hexamer complex, in gel filtration chromatography RuvA domain III and RuvB elute as a complex with a 1:1 stoichiometry (36). Such a complex was proposed as a preloading complex to recruit RuvB subunits and facilitate their assembly on the DNA. Thus, the RuvA/RuvB system exhibits strong mechanistic parallels with the results presented here for TrwA/TrwB. Some hexameric helicases, such as bovine papillomavirus replication initiator protein E1, also form a complex with the accessory protein E2 (37). Protein E2 determines the DNA binding specificity of the E1 hexamer at the origin of replication, forming a pre-replication complex. A model has also been proposed by which, following this association, the recruitment of additional E1 monomers would lead to the assembly of the E1 hexamer at the viral origin (30).


Figure 7
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  		FIGURE 7.
A model for TrwB assembly on DNA mediated by TrwA. A, RuvA protein (in red) is bound as a double tetramer to a square planar structure on the DNA, called Holliday junction. Each RuvA tetramer facilitates the assembly of one hexameric RuvB motor, represented in green. B, TrwA (in red) is represented also as a double tetramer bound to an inverted repeat called IR4, its specific recognition sequence at oriT, forming a hairpin in the figure. TrwB monomers anchored in the inner membrane are represented in green. OM and IM represent the outer and inner membrane, respectively. By analogy to the RuvA/RuvB paradigm, two TrwA tetramers have been drawn to maintain the structure on the DNA, but only one hexamer assembles, because it requires a ssDNA scaffold to oligomerize. In both cases, oligomerization and assembly of the hexameric motor on the DNA seems to be mediated by its interaction with TrwA or RuvA, tetrameric proteins, which previously bind specifically to the DNA. RuvB couples ATP hydrolysis to dsDNA pumping, whereas in bacterial conjugation ssDNA is the transported substrate.

 
We can extrapolate this model to the TrwA/TrwB system to explain TrwA/TrwB interactions in the DNA translocation process toward the membrane (see Fig. 7). TrwA, as a tetramer bound to a specific inverted repeat in the oriT (IR4), would interact through the C-terminal domain with a TrwB monomer anchored in the inner membrane, promoting its oligomerization. The inverted repeat IR4, which is specifically recognized by TrwA, probably forms also a cruciform on the DNA. To summarize, the biochemical analysis presented here shows that TrwA, as RuvA, facilitates oligomerization and loading of TrwB on the DNA, modulating its activity. It is tempting to speculate that a universal mechanism to load hexameric motors onto DNA has been conserved in evolution. The model also explains the coupling role proposed for TrwB, connecting the relaxosome to the T4SS, in the membrane. Once the cleavage reaction is carried out by the relaxase TrwC, TrwB would couple the energy released from ATP hydrolysis to DNA pumping, as previously proposed (19).


   FOOTNOTES
 
* This work was supported in part by Grant BFU2005-02718 (Spanish Ministry of Education) (to E. C.) and Grants BFU2005-03477/BMC (Spanish Ministry of Education) and LSHM-CT-2005_019023 (European VI Framework Program) (to F. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by a fellowship from the Basque Government. Back

2 To whom correspondence may be addressed. E-mail: delacruz{at}unican.es. 3 To whom correspondence may be addressed. E-mail: cabezone{at}unican.es.

4 The abbreviations used are: oriT, origin of transfer; PMSF, phenylmethylsulfonyl fluoride; Pipes, 1,4-piperazinediethanesulfonic acid; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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