JOURNAL OF BACTERIOLOGY, May 2010, p. 2315–2323 Vol. 192, No. 90021-9193/10/$12.00 doi:10.1128/JB.01384-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Biochemical Dissection of the ATPase TraB, the VirB4 Homologue ofthe Escherichia coli pKM101 Conjugation Machinery�†‡

Eric Durand,§ Clasien Oomen, and Gabriel Waksman*Institute of Structural and Molecular Biology, UCL/Birkbeck, Malet Street, London WC1E 7HX, United Kingdom

Received 21 October 2009/Accepted 11 February 2010

Type IV secretion (T4S) systems are involved in several secretion processes, including secretion of virulencefactors, such as toxins or transforming molecules, or bacterial conjugation whereby two mating bacteriaexchange genetic material. T4S systems are generally composed of 12 protein components, three of which,termed VirB4, VirB11, and VirD4, are ATPases. VirB4 is the largest protein of the T4S system, is known to playa central role, and interacts with many other T4S system proteins. In this study, we have biochemicallycharacterized the protein TraB, a VirB4 homologue from the pKM101 conjugation T4S system. We demon-strated that TraB is a modular protein, composed of two domains, both able to bind DNA in a non-sequence-specific manner. Surprisingly, both TraB N- and C-terminal domains can bind ATP, revealing a new degen-erated nucleotide-binding site in the TraB N-terminal domain. TraB purified from the membrane forms stabledimers and is unable to hydrolyze ATP while, when purified from the soluble fraction, TraB can form hexamerscapable of hydrolyzing ATP. Remarkably, both the N- and C-terminal domains display ATP-hydrolyzingactivity. These properties define a new class of VirB4 proteins.

The type IV secretion (T4S) systems are widely distributedamong the Gram-negative and -positive bacteria. T4S systemsexport proteins and DNA-protein complexes across the bacte-rial cell envelope to other bacteria or eukaryotic cells, gener-ally through a process requiring direct cell-to-cell contact (10,11, 16). T4S systems have been grouped according to sequencerelatedness of machine components, with systems homologousto the archetypal VirB/VirD4 T4S system of Agrobacteriumtumefaciens being classified as type IVA and those related tothe Dot/Icm T4S system of Legionella pneumophila being clas-sified as type IVB (12). T4S systems fulfill a wide variety offunctions, such as mediating the conjugative transfer of plas-mids and other mobile DNA elements to bacterial recipientcells or delivering protein or DNA substrates to eukaryoticcells. Another kind of T4S system-related process is DNArelease or uptake, whereby DNA substrates are exchangedwith the extracellular milieu (8, 16).

T4S-related machineries are used by several plant andhuman pathogens for the purpose of delivering virulenceeffectors to eukaryotic cell targets. Such pathogens includeextracellular organisms such as A. tumefaciens, which is thecausative agent of crown gall disease in plants, Bordetella per-tussis, which is the agent responsible for whooping cough inchildren, and Helicobacter pylori, which is responsible for gas-tric ulcers and stomach cancer (3, 6, 13, 34). In addition, there

are intracellular bacterial pathogens using T4S systems fortheir virulence, such as Brucella suis, the causative agent ofbrucellosis, and L. pneumoniae, the causative agent of Legion-naires’ disease (5, 30).

T4S systems are generally composed of 12 protein compo-nents forming a macromolecular assembly inserted into thebacterial cell envelope. These proteins are named VirB1 toVirB11 and VirD4, based on the widely used nomenclature ofthe model system, the A. tumefaciens VirB/D4 T4S system (16).Three putative ATPases are key components of the T4S sys-tem: VirD4, VirB11, and VirB4. VirB4 proteins are the largestand most evolutionarily conserved proteins in T4S systems(15). VirB4 proteins are suggested to be located in the innermembrane, either directly and/or indirectly through their in-teractions with other components of the T4S system (14, 18,31). An important feature of VirB4 proteins is the presence ofWalker A and Walker B motifs characteristic of ATPases (29).But until very recently no nucleoside triphosphatase (NTPase)activity had been demonstrated for any VirB4 homologue.However, the VirB4 homologue of plasmid R388, the proteinTrwK, has now been shown to possess an ATPase activity (2).Very little structural information is available for the VirB4subunit family. Recently, a bioinformatics model based on thestructural similarities between the Agrobacterium VirB4 C ter-minus and TrwB (VirD4 homolog) proposed that the VirB4 Cterminus forms a discrete domain that assembles as a homo-hexameric ring (26), much like VirB11 and VirD4 (19, 33).

Here, we report a comprehensive biochemical study of theTraB protein, the VirB4 homologue from the pKM101 conju-gation machinery. Our results suggest that TraB exists undertwo forms: a dimeric membrane form and a primarily hexa-meric soluble form. Both bind DNA nonspecifically and alsoATP; however, only the hexameric form hydrolyzes ATP. TraBhas a clear modular structure composed of two large contigu-ous domains that split the protein roughly in two. While onlythe C-terminal domain was known to contain a nucleotide-

* Corresponding author. Mailing address: Institute of Structural andMolecular Biology at UCL and Birkbeck, Malet Street, London WC1E7HX, United Kingdom. Phone: 44 207 631 6833. Fax: 44 207 631 6803.E-mail: g.waksman@ucl.ac.uk.

§ Present address: LISM (IBSM), 31 Chemin Joseph Aiguier, 31402Marseilles, France.

† Supplemental material for this article may be found at http://jb.asm.org/.

� Published ahead of print on 19 February 2010.‡ The authors have paid a fee to allow immediate free access to

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binding site, we establish that both domains can bind andhydrolyze ATP. Also, both cooperate to bind DNA. Our studyalso reveals a complex interplay between the two domains. Ourresults provide crucial but singular insights that suggest previ-ously unsuspected properties of a family of proteins that playcritical functions in T4S systems.

MATERIALS AND METHODS

Cloning of TraB and TraB domains. The full-length traB gene (traBFL; aminoacids [aa] 1 to 866), along with the region encoding the N-terminal domain(traBNT; aa 1 to 442) and the C-terminal domain (traBCT; aa 448 to 848) werePCR-amplified from the pKM101 plasmid and cloned into the pET151/D-TOPOvector (Invitrogen) following standard TOPO cloning protocols. Consequently,all three constructs allow the expression of N-terminally His6-tagged recombi-nant proteins, named TraBFL, TraBNT, and TraBCT. After DNA sequencing tocheck that the sequences did not contain any mutations, the four plasmids weretransformed by heat shock in chemically competent Escherichia coli BL21 Star(DE3) cells (Invitrogen) for large-scale production of the recombinant proteins.

The Walker A mutants were generated by PCR amplification of the aboveplasmids using primers carrying the appropriate mutation. To mutate the firstnucleotide-binding site (NBD1) of TraB, we used NT-RA1 (5�-TTTTTCAAGCTGGATGGCGCAACACATGACTGCGCATCAGATCGG-3�) and NT-RA2 (5�-TCTGATGCGCAGTCATGTGTTGCGCCATCCAGCTTGAAAAAAGCC-3�). This mutates residue Arg53 into Ala (mutated residues are inboldface). To mutate the second nucleotide-binding site (NBD2), we usedCT-KA1 (5�-GGTATGTCGGGGGAAGGTGCGACCACGCTGCTTAACTTCCTGCTGGC-3�) and CT-KA2 (5�-GAAGTTAAGCAGCGTGGTCGCACCTTCCCCCGACATACCCGTTATTAACGC-3�). This mutates residue Lys504 intoAla (mutated residues are in boldface). After PCR, amplified plasmids weresubjected to DpnI digestion in order to remove the original wild-type plasmid.After purification using an Extract-II kit (Nalgene), plasmids were trans-formed by heat shock in chemically competent BL21 Star (DE3) cells (In-vitrogen). The presence of the mutation was verified by DNA sequencing ofthe isolated plasmids.

Production and purification of recombinant proteins. E. coli strain BL21 Star(DE3) (Invitrogen) containing one of the recombinant pET151 plasmids (forTraBFL, TraBNT, or TraBCT) was grown at 37°C in Terrific Broth (Merck)supplemented with 100 �g/ml ampicillin (Sigma-Aldrich) until the culture reachedan A600 of 1.2. Cultures were then shifted to 16°C for 1 h before isopropyl-�-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, andgrowth was then continued for 15 h at 16°C. Cells were harvested by centrifugation,resuspended in 20 mM Tris-HCl (pH 7.5), and stored at �20°C.

All purification steps were carried out at 4°C. TraBCT is soluble while TraBFL

and TraBNT partition between soluble forms in the cytoplasm and membraneforms in the inner membrane. TraBCT and the soluble forms of TraBFL andTraBNT were purified from cytoplasmic extracts as follows. The cells were de-frosted and resuspended in a buffer (3 ml per g of cell paste) containing 20 mMTris-HCl, pH 7.5, 300 mM NaCl, 1 mM �-mercaptoethanol (�ME), and onetablet of EDTA-free protease inhibitor cocktail (Roche). After cells were brokenby two passages through an EmulsiFlex-C5 homogenizer and DNA was frag-mented by sonication, the lysate was clarified by centrifugation at 18,000 rpm for45 min in a Sorvall SS-34 rotor. The clarified lysate was loaded onto a HisTrapHP (high-performance) 5-ml column (GE Healthcare) equilibrated in buffer Asol

(20 mM Tris-HCl [pH 7.5], 300 mM NaCl, 1 mM �ME; sol indicates buffer usedto purify the soluble forms of TraB proteins) plus 4% of buffer Bsol (20 mMTris-HCl [pH 7.5], 300 mM NaCl, 1 mM �ME, 500 mM imidazole). The columnwas then washed with 100 ml of buffer Asol plus 8% buffer Bsol. Finally theproteins still bound to the column were eluted in a gradient from 8% to 100% ofbuffer Bsol in 100 ml. Eluted fractions containing the protein of interest werepooled and concentrated in less than 4 ml before being loaded onto a HiPrep16/60 Sephacryl S-300 HR column (Amersham) equilibrated in the gel filtration(GF) buffer GFsol containing 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 1 mM�ME or in a buffer with acetate, termed GFacetate-sol and containing 50 mMHEPES-NaOH (pH 7.0), 75 mM potassium acetate, 2 mM magnesium acetate,10% (wt/vol) glycerol, and 0.1 mM EDTA (see also the Results and Discussionsection for the naming of the gel filtration buffers). The protein of interest elutedas a single peak. Fractions under this peak were pooled.

TraBFL and TraBNT were also purified from the membranes. When proteinswere purified from membrane extracts, the following protocol was applied. Thecells were defrosted and resuspended in a buffer (3 ml per g of cell paste)containing 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM �ME, and one tablet

of EDTA-free protease inhibitor cocktail (Roche). After cells were broken bytwo passages through an EmulsiFlex-C5 homogenizer and DNA was fragmentedby sonication, unbroken cells were removed by centrifugation at 14,000 rpm for10 min in a Sorvall SS-34 rotor. Total membranes were pelleted by ultracentrif-ugation (45 min at 100,000 � g at 4°C) and resuspended in buffer EB (20 mMTris-HCl [pH 7.5], 50 mM NaCl, 1 mM �ME, 1% [vol/vol] Triton X-100)supplemented with one tablet of EDTA-free protease inhibitor cocktail (Roche).Membrane-embedded proteins were extracted during 1 h at 4°C. The membraneextract was further clarified by ultracentrifugation (30 min at 100,000 � g at 4°C).Triton X-100 was used only for extraction; then it was replaced by the hydroge-nated Triton X-100 [Triton X-100(H); Calbiochem] that does not absorb UVlight. We further used a concentration of 0.01% Triton X-100(H) (0.16 mM)because this is below the critical micelle concentration (CMC) of the detergent(0.2 to 0.9 mM), thus avoiding the formation of detergent micelles. The clearedextract was loaded onto a HisTrap HP 5-ml column (GE Healthcare) equili-brated in buffer Amb [20 mM Tris-HCl (pH 7.5(, 300 mM NaCl, 1 mM �ME,0.01% Triton X-100(H); mb indicates the buffer used to purify the membraneforms of TraB proteins] plus 4% of buffer Bmb [20 mM Tris-HCl (pH 7.5), 300mM NaCl, 1 mM �ME, 0.01% Triton X-100(H), 500 mM imidazole]. The columnwas then washed with 100 ml of buffer Amb plus 6% buffer Bmb. Finally, theproteins still bound to the column were eluted in a gradient from 6% to 100% ofbuffer Bmb in 100 ml. Eluted fractions containing either TraBFL or TraBNT werepooled and concentrated in less than 4 ml before being loaded onto a HiPrep16/60 Sephacryl S-300 HR column (Amersham) equilibrated in either bufferGFmb containing 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM �ME, and0.01% Triton X-100(H) or buffer GFacetate-mb containing 50 mM HEPES-NaOH(pH 7.0), 75 mM potassium acetate, 2 mM magnesium acetate, 10% (wt/vol)glycerol, 0.1 mM EDTA, and 0.01% Triton X-100(H) (see also the Results andDiscussion section for the naming of the gel filtration buffers). The proteinsTraBFL and TraBNT both eluted as a single peak. Fractions under this peak werepooled.

The apparent molecular mass of proteins eluted from the gel filtration columnwas deduced from a calibration carried out with low- and high-molecular-weightcalibration kits (Amersham Biosciences). In addition to dynamic light scattering(DLS; described below), the molecular weights of the various multimeric formswere assessed using 3 to 12% or 4 to 16% blue native (BN)-PAGE (Invitrogen).Determination of protein concentration was carried out by using either thetheoretical absorption coefficients (mg/ml � cm) at 280 nm as obtained with theprogram ProtParam at the EXPASY server (available on the World Wide Web atwww.expasy.ch/tools) or a Bio-Rad protein assay reagent (Bio-Rad).

ATPase assays. Two sets of assays were carried out to assess the ATPaseactivity of all purified proteins. For the first set of assays, we used the proteins inGFsol for the soluble forms or those in GFmb for the membrane forms. TheATPase assays were carried out using an Innova Bioscience kit, which requiredthat the assay be performed in a final buffer containing 50 mM Tris-HCl (pH7.4), 25 mM NaCl, 2.5 mM MgCl2, and 0.5 mM ATP (the enzyme concentrationwas 70 �M [in monomer equivalent]).

For the second set of ATPase assays, we used the proteins in GFacetate-sol forthe soluble forms and those in GFacetate-mb for the membrane forms. ATPaseactivity was monitored using a coupled-enzyme assay (24). A total of 44 �l ofTraBFL (3.3 �M), TraBNT (7.5 �M), and TraBCT (10.3 �M) in GFacetate-sol (forthe soluble forms of the three proteins) or in GFacetate-mb (for the membraneforms of TraBFL and TraBNT) was incubated in 400 �l of ATP assay buffer,consisting of 50 mM PIPES [piperazine-N,N�-bis(2-ethanesulfonic acid)]-NaOH(pH 6.45), 75 mM potassium acetate, 5% (wt/vol) glycerol, 10 mM magnesiumacetate, 1 mM potassium chloride, 1 mM dithiothreitol, 0.1 mM EDTA, 0.5 mMphosphoenolpyruvate, 0.25 mM NADH, 60 �g/ml pyruvate kinase, 60 �g/mllactate dehydrogenase, and 0.0625 to 10 mM ATP. The reaction mixtures werepreincubated at 37°C for 2 min, after which the ATPase assay was started by theaddition of various concentrations of ATP. Activity was measured by the de-crease in NADH absorbance at 340 nm for 15 min at 37°C in a UV-visiblespectrophotometer (CARY 3; Varian) and the slopes (absorbance/min) werecalculated by the program Kinetics-CARY. All the data sets were fitted to aMichaelis-Menten equation:

Vi � Vmax � [S/(Km � S)] (1)

where Vi is the initial velocity or specific ATPase activity (mol of ATP hydrolyzedper min per mol of enzyme [monomer equivalent]), Vmax is the maximum velocityof the enzyme at substrate saturation, S represents the concentration of ATP(mM), and Km is the substrate concentration at which the initial velocity reacheshalf of its maximum value (Vmax/2) and represents the Michaelis constant. To

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better evaluate the enzymatic parameters of the various TraB domains, we useda Lineweaver-Burk plot and fitted the data sets to a linear regression:

1/Vi � Km/Vmax � (1/S) � (1/Vmax) (2)

Note that adding 0.01% Triton to the soluble form of TraBFL did not affect itsATPase activity; thus, 0.01% Triton does not affect ATPase activity of TraBproteins.

DLS. Dynamic light-scattering experiments were performed with a DynaPro-801 (Protein Solutions) at room temperature. All samples were filtered prior tothe measurements (Millex syringe filters; 0.22-�m pore size; Millipore Corp.).The hydrodynamic radius was deduced from translational diffusion coefficientsusing the Stokes-Einstein equation. Diffusion coefficients were inferred from theanalysis of the decay of the scattered intensity autocorrelation function. Allcalculations were performed using the software provided by the manufacturer(Dynamics, version 5.25.44).

DNA mobility assay. Proteins (in GFsol or GFmb) at various concentrationsand DNA (pKM101 plasmid or PCR product) were mixed and incubated for 10min at room temperature. A small volume of loading buffer (0.005% bromophe-nol blue–20% [wt/vol] sucrose) was added to the sample before it was loaded ona native agarose gel prepared using 0.3% or 0.4% agarose dissolved in SYBRSafe DNA gel stain (Invitrogen) in 0.5� TBE (Tris-Borate-EDTA) buffer tovisualize the free DNA and the protein-DNA complex bands. Gels were elec-trophoresed at 100 V for 90 min to 2 h at room temperature in 0.5� TBE buffer.Free DNA and DNA-protein complex bands were visualized under a UV lamp.

Limited trypsin proteolysis. Protein and DNA (pKM101 plasmid) were pre-incubated for 10 min on ice in buffer GFmb or GFsol prior to the addition oftrypsin (0.3 �g/ml). The reactions were allowed to proceed on ice for 60 min. Atvarious time points (0, 15, 30, 45, and 60 min), an aliquot was removed from thereaction mixture, and NuPAGE lithium dodecyl sulfate (LDS; 4�) sample buffer(Invitrogen) was added. Samples were boiled for 10 min and then loaded on aNu-PAGE (4 to 12%) gel (Invitrogen) and electrophoresed for 35 min at 200 Vin NuPAGE morpholinepropanesulfonic acid (MOPS)-SDS running buffer (In-vitrogen). After electrophoresis the gel was stained with SimplyBlue SafeStain(Invitrogen).

Fluorescence measurements. The fluorescent ATP nucleotide analogue 3�(2�)-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) was purchased from MolecularProbes. Fluorescence measurements were performed on a Hitachi F2500 Fluo-rescence Spectrophotometer, and all the data were processed using the softwareFL Solutions F-2500. The excitation wavelength was set at 410 nm, and theemission wavelength was scanned in the 470- and 650-nm range. TNP-ATPbinding was calculated from the fluorescence maxima determined graphically.The temperature of the sample was maintained at 20°C by circulating thermo-statically controlled water through the cuvette holder. For determination of thedissociation constant of TNP-ATP (Kd

TNP-ATP), 1 �M (monomer concentration)protein solutions (in GFsol or GFmb) were titrated with TNP-ATP. The Kd valuesfor MgATP (Kd

ATP) were determined by displacement of protein-bound TNP-ATP. Protein solutions (1 �M) were incubated for 20 s with either 30 �M (forTraBFL) or 15 �M (for TraBNT and TraBCT) TNP-ATP. MgATP aliquots werethen added from 0.1 M stock solution incrementally, and fluorescence wasmeasured after incubation for 20 s. All spectra were corrected for buffer fluo-rescence and for dilution (never exceeding 5% of the original volume). Titrationcurve fitting was accomplished using ProFit for Mac OS X, version 6.1.4 (Quan-tum Soft) with the following quadratic equation in the case of increasing TNP-ATP fluorescence (21):

�F � �Fmin � {(�Fmax � �Fmin)[(Et � L � KdTNP-ATP)

� ((Et � L � KdTNP-ATP)2 � 4EtL)1/2]}/2Et (3)

where �F represents the relative fluorescence intensity, �Fmin is the relativefluorescence intensity at the start of the titration, �Fmax is the relative fluores-cence intensity at a saturating concentration of TNP-ATP (L), Et is the totalconcentration of protein (monomer equivalent), and Kd

TNP-ATP is the apparentdissociation constant of the protein–TNP-ATP complex.

In the case of displacement of bound TNP-ATP by ATP, the following qua-dratic equation was used (21):

�F � �Fmax � {(�Fmax � �Fmin)[(Et � L � K0.5)

� ((Et � L � K0.5)2 � 4EtL)1/2]}/2Et (4)

where �Fmax is the relative fluorescence intensity at the start of the titration, and�Fmin is the relative fluorescence intensity at saturating concentration ofMgATP, K0.5 represents here the amount of MgATP necessary to displace half

the amount of bound TNP-ATP, and L represents the MgATP concentration.We then used the K0.5 value obtained from the displacement experiments and thefollowing equation to calculate the apparent dissociation constant of the protein-MgATP complex (Kd

ATP):

KdATP � K0.5/[1 � (L/Kd

TNP-ATP)] (5)

where L represents the TNP-ATP concentration at the start of the titration.

RESULTS AND DISCUSSION

Identification and production of TraB domains. TraB, likeother VirB4 proteins, contains a nucleoside triphosphate (NTP)-binding site encompassing the Walker A and Walker B motifslocated in the second half of the protein sequence (Fig. 1A,NBD). This C-terminal region (residues 448 to 848) is also themost conserved among VirB4 proteins and, as already sug-gested by Middleton et al. (26) might be similar in structure tothe soluble domain of TrwB, a VirD4 homolog (19; see alsoFig. S1 in the supplemental material). Therefore, we decidedto clone and express individually the N- and C-terminal do-mains of TraB, together with the full-length protein (Fig. 1 andMaterials and Methods). The three proteins were expressedin E. coli as N-terminally His6-tagged recombinant proteins.The C-terminal domain of TraB (TraBCT) was purified fromthe soluble fraction (see Materials and Methods). However,because a distinct single transmembrane domain (TM) inthe N-terminal region of TraB is predicted (http://www.sbc.su.se/miklos/DAS/), the full-length TraB (TraBFL) and the N-terminal domain of TraB (TraBNT) were initially purified fromthe membrane fraction. However, we noticed that both TraBFL

and TraBNT partitioned equally in the soluble and membranefractions, and thus soluble forms of both TraBFL and TraBNT

FIG. 1. Isolation of TraB domains. (A) Schematic representationof the domain structure of TraB. N, N terminus; C, C terminus; TM,putative transmembrane domain; NBD, NTP binding domain; TraBFL,residues 1 to 866; TraBNT, residues 1 to 442; TraBCT, residues 448 to848. (B) SDS-NuPAGE 4 to 12% showing the purified proteins aftergel filtration. Lane 1, TraBFL (102 kDa); lane 2, TraBNT (55 kDa); andlane 3, TraBCT (48.8 kDa). Molecular mass markers are indicated onthe left side of the gel (kDa).

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were also purified (see Materials and Methods). TraBCT wasnever found in the membrane fraction. All proteins were pu-rified to homogeneity using the same two-step purificationstrategy (Fig. 1B and Materials and Methods).

Oligomerization state of TraBFL, TraBNT, and TraBCT. InSDS-PAGE the proteins migrate at their expected molecularmasses: 102 kDa for TraBFL, 55 kDa for TraBNT, and 48.8 kDafor TraBCT (Fig. 1B). However, gel filtration analysis indicatedthat the proteins are oligomeric. Oligomerization was testedunder two buffer conditions established at the final gel filtra-tion stage of the purification. These conditions together withthe cell compartment from which the proteins originate (cy-tosol or membrane) are referred to in Materials and Methodsand thereafter as GFsol, GFacetate-sol, GFmb, and GFacetate-mb,where sol and mb refer to the soluble cytosolic and membraneforms of the proteins, respectively, and acetate indicates thatthe buffer contains acetate. The membrane form of TraBFL inGFmb is primarily dimeric, as assessed by BN-PAGE and gelfiltration (see Fig. S2A in the supplemental material) althoughhigher-order oligomers (tetramers and hexamers) are also ob-served using BN-PAGE. The fractions containing the dimersof membrane-extracted TraBFL were selected. These dimersare stable over time and were used for subsequent studies ofthe membrane form of TraBFL. TraBNT purified from themembrane fraction is overwhelmingly dimeric, in GFmb, al-though large aggregates eluting in the void volume are ob-served, indicating a tendency of the protein to aggregate (seeFig. S2B). The oligomerization state of TraBFL or TraBNT inGFacetate-mb was not investigated.

TraBCT and the soluble forms of TraBFL and TraBNT wereall dimeric in GFsol (see results for TraBCT in Fig. S2C in thesupplemental material). All three were, however, hexameric inGFacetate-sol, as assessed using gel filtration (data not shown)and DLS (Table 1). Thus, we conclude that the soluble formsof TraBCT, TraBFL, and TraBNT transition between at leasttwo forms, dimeric and hexameric, which are in a dynamicequilibrium where one form is favored over the other depend-ing on solution conditions, notably the presence or absence ofacetate ions. Whether the membrane forms of TraBFL andTraBNT also transition between two oligomeric states remainsto be investigated. It is, however, interesting that these proteins

do not display ATPase activities even in the presence of ace-tate ions (see “ATP hydrolysis of TraB proteins” below), sug-gesting that dimeric TraBFL and TraBNT might not be able totransition to higher oligomeric forms. This could be caused bythe presence of detergent in the GFacetate-mb buffer, but it wasshown that detergent does not disrupt ATPase activities of thesoluble TraB protein forms (see “ATP hydrolysis of TraB pro-teins” below).

These results contrast with those obtained on the full-lengthTrwK protein, the VirB4 homologue in the R388 plasmid sys-tem, for which no membrane form has been identified. TrwKwas found to be soluble and primarily monomeric although aminor hexameric form accounting for 5% of the total specieswas ascribed as the ATP-hydrolyzing form (2). Also, no TrwKfragment has been studied.

Because TrwK has already been the subject of extensivestudies, we decided to focus subsequent work on TraB andTraB fragments primarily (but not exclusively; see ATPaseassay results below), specifically, on the dimeric, membraneforms of TraBFL and TraBNT (purified in GFmb) and on thedimeric soluble form of TraBCT (purified in GFsol).

Interaction of TraB-derived constructs with DNA. BecauseTraB is a component of a T4S system transporting DNA, weinvestigated whether TraB could bind DNA. First, we showedthat TraBFL (membrane form in GFmb) is protected fromproteolysis degradation after being incubated in the presenceof the pKM101 DNA (Fig. 2A). This result suggested thatTraB was able to interact with the pKM101 DNA. In a secondapproach, we demonstrated that TraBFL could shift the migra-tion of the pKM101 DNA on a native agarose gel in a concen-tration-dependent manner (results not shown). However, thisinteraction is not DNA sequence specific since TraBFL couldalso shift the migration of a random PCR product (Fig. 2B).We conclude that TraBFL can bind DNA nonspecifically.Then, we asked which of the two TraB domains is responsiblefor DNA binding. Interestingly, both the N-terminal (TraBNT;membrane form in GFmb) and C-terminal (TraBCT; dimericsoluble form in GFsol) domains of TraB were able to bindeither the pKM101 DNA (results not shown) or a random PCRproduct (Fig. 2B). In this assay, an unrelated protein (bovineserum albumin [BSA]) was unable to bind the PCR product(Fig. 2B). In conclusion, we demonstrated that TraB can bindDNA in a non-sequence-specific manner and that both theN-terminal and C-terminal domains are involved in binding.Interestingly, although the end shift positions are similar for allthree proteins, saturation occurs more rapidly for TraBCT thanfor either TraBFL or TraBNT. This indicates an influence ofTraBNT on DNA binding of TraBCT.

TraB-derived proteins can bind ATP. Until now, only oneconserved nucleotide-binding site has been identified in TraB,and the site is located in its C-terminal domain (Fig. 1). Thisprompted us to investigate whether TraBCT could bind ATP.We used fluorescence spectroscopy to monitor the interactionof the fluorescent ATP analogue TNP-ATP with the protein.Upon protein binding, the fluorescence emission intensity ofTNP-ATP increases considerably, with the absolute magnitudedependent on the specific protein environment; thus, it hasbeen widely used to characterize ATP binding by a number ofproteins (9, 25). As expected, we showed that TraBCT (solubledimeric form in GFsol) was able to bind TNP-ATP, as demon-

TABLE 1. DLS data of the soluble TraB-derivedproteins in GFacetate

Protein (NBD1and/or NBD2)a

MMCalc(kDa)b

HexamerMMCalc(kDa)c

MMDLS(kDa)d

Radius(nm)e

Polydispersity(nm %�)

TraBFL (WT/WT) 102.6 615.6 593.0 9.1 3.5 (38.1)TraBFL (WT/KA) 102.6 615.6 603.4 9.2 3.1 (33.6)TraBFL (RA/WT) 102.6 615.6 666.9 9.6 3.8 (39.6)TraBFL (RA/KA) 102.6 615.6TraBNT (WT) 54.6 327.6 365.6 7.4 2.9 (39.0)TraBNT (RA) 54.6 327.6TraBCT (WT) 48.8 292.8 348.5 7.3 2.3 (32.4)TraBCT (KA) 48.8 292.8 353.3 6.9 2.3 (24.2)

a The presence or absence of nucleotide-binding domain mutation(s) is shownin parentheses for the full-length TraB and fragments. WT, wild type; RA, R53Amutation in NBD1; KA, K504A mutation NBD2.

b MMCalc, theoretical molecular mass calculated from amino acid sequence.c The hexamer molecular mass was calculated as MMCalc � 6.d Molecular mass experimentally determined by DLS.e Estimation of the hydrodynamic radius of the particle.

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strated by the remarkable enhancement of the emission inten-sity of TNP-ATP in the presence of TraBCT (Fig. 3A) togetherwith a blue shift (from 552 to 546 nm) of the wavelength ofmaximal emission. Moreover, bound TNP-ATP could be dis-lodged from its binding site, at least partially, by excess ATP(Fig. 3B), indicating that the reported TNP-ATP-enhancedemission intensity is caused by binding to a bona fide ATP-binding site (21, 32).

TraBCT represents just one half of the full-length TraB(TraBFL). So, we wanted to check if the rest of the protein couldinfluence the ATP-binding property of the C-terminal domainof TraB. We used fluorescence spectroscopy to monitor theinteraction of TNP-ATP with TraBFL and TraBNT, both puri-fied from the membrane fraction (i.e., in GFmb). Similarly toTraBCT, TraBFL was able to bind TNP-ATP (Fig. 3A), albeitwith a slightly different absolute magnitude that could indicatea different protein environment around the bound TNP-ATP.Furthermore, TNP-ATP binding was efficiently competed byadding an excess of ATP (Fig. 3A). Surprisingly, TraBNT alsobinds TNP-ATP, a binding reaction that was also competed byan excess of ATP (Fig. 3A). The latter result was unexpected,given that the only reported nucleotide-binding site in VirB4proteins is located in the C-terminal half (Fig. 1).

The three TraB proteins (the soluble dimeric TraBCT inGFsol and the membrane TraBFL and TraBNT in GFmb) werenext titrated with increasing amounts of TNP-ATP, and theresults were fitted to equation 3 of Materials and Methods(Fig. 3B), from which apparent dissociation constants for TNP-ATP (Kd

TNP-ATP) could be derived. Equation 3 is derived froma single-site binding model, and the protein concentration is inmonomer equivalent. The values for Kd

TNP-ATP were 6.91 �0.79 �M for TraBFL, 1.22 � 0.30 �M for TraBNT, and 0.51 �0.08 �M for TraBCT (Table 2). Thus, as observed for DNAbinding, we observe a functional interaction between the twodomains which, when they come together in the full-lengthprotein, results in enhanced binding affinity for TNP-ATP.

Bioinformatics analysis of TraBNT and identification of itsATP-binding site. In order to explain the ATP-binding prop-

erty of TraBNT, we analyzed the protein sequence of TraB.Using BLAST or ScanProsite (http://www.expasy.ch/tools/blast/;http://www.expasy.ch/tools/scanprosite/), we could not detect asecond binding site in the N-terminal domain of TraB. Thus,we sought to compare TraB with known ATP-binding proteinswith two nucleotide-binding sites. ATP-binding cassette (ABC)transporters have two nucleotide-binding sites (28), but both ofthem are highly conserved. Conversely, the translocon proteinSecA has two nucleotide-binding sites (see Fig. S3A in thesupplemental material), a high-affinity site in the N-terminaldomain (NBD1) and a low-affinity site in the C-terminal do-main (NBD2) (27); the low-affinity site is far less conserved inits amino acids sequence than the high-affinity site. We thusperformed a sequence alignment between the N-terminal do-main of TraB and the C-terminal domain of SecA (see Fig.S3B). Surprisingly, the two domains exhibit a high degree ofsequence identity (28%) (see Fig. S3B). Moreover, two regionsin the TraB N-terminal domain align with the sequencesknown to form the Walker A and B motifs in the C-terminaldomain of SecA (see Fig. S3B). Notably, key residues (GRXTXD) in the Walker A motif of SecA are conserved in TraBNT

(20). We concluded that, like the SecA C-terminal domain, theN-terminal domain of TraB also contains a poorly conservednucleotide-binding site. We then asked if this new feature inthe N-terminal domain of TraB was conserved among theVirB4 protein family. As shown in Fig. 4, the nucleotide-bind-ing site in the N-terminal domain of TraB seems to be poorlyconserved among VirB4 proteins. Indeed, just 3 out of the 38VirB4 homologues aligned, including TraB, present a motifsimilar to the SecA low-affinity NTP-binding site. Thus, wehave identified a new ATP-binding site in the N-terminal do-main of TraB that defines a new class of VirB4 proteins (Fig.4; see also and Fig. S3C and S4 in the supplemental material).

ATP hydrolysis of TraB proteins. Recently, the first demon-stration of an ATPase activity for a VirB4 homolog was de-scribed for the protein TrwK from the plasmid R388 T4Ssystem conjugation system (2). ATPase assays of TraB andTraB fragments were carried out under two sets of conditions

FIG. 2. Interaction between TraB and DNA. (A) Limited proteolysis of TraBFL in the absence or presence of DNA. TraBFL purified in GFmb

(23.1 �g) and the pKM101 plasmid (1.94 �g) were preincubated together in buffer GFmb (see Materials and Methods) for 10 min on ice prior tothe addition of trypsin (0.3 �g/ml). The reactions were allowed to proceed on ice for the indicated periods of time. The reactions were carried outin the presence (�) or absence (�) of the pKM101 plasmid. The arrow indicates TraBFL. Molecular mass markers are indicated on the left sideof the gel. (B) Interaction of TraB and TraB fragments with DNA. Native 0.3% agarose gel shift electrophoresis was used to analyze the associationof the different TraB domains with a random PCR product DNA: TraBFL purified in GFmb (a), TraBNT purified in GFmb (b), TraBCT purified inGFsol (c), and BSA (d). The amounts of protein (nmol) are indicated above each gel, and the amount of DNA was 350 ng in each reaction mixture.The arrows on each gel indicate the unshifted position of the PCR product DNA.

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(see Materials and Methods), i.e., in a buffer similar to GFsol/GFmb and in a buffer similar to GFacetate-sol/GFacetate-mb.

In GFacetate-sol, TraBCT was found to hydrolyze ATP. Undersuch conditions, it is also hexameric (see above). TraBCT ki-

netic parameters for ATP hydrolysis were determined by ana-lyzing the effect of ATP concentration on ATPase activity rates(Fig. 5). The data were fitted to the Michaelis-Menten equa-tion 1, from which we calculated the kinetic parameters.

FIG. 3. ATP-binding to TraB and TraB fragments. (A) TraB and TraB fragments bind TNP-ATP. The fluorescent ATP analogue TNP-ATPdisplayed enhanced fluorescence upon binding by TraBFL purified in GFmb (a), TraBNT purified in GFmb (b), and TraBCT purified in GFsol (c).Fluorescence spectra 1 to 4 were taken from the following samples: spectrum 1, protein (5 �M); spectrum 2, TNP-ATP (23 �M); spectrum 3,protein (5 �M) plus TNP-ATP (23 �M); spectrum 4, protein (5 �M) plus TNP-ATP (23 �M) in the presence of ATP (0.5 mM). Fluorescenceintensities are expressed in arbitrary units. These curves are the averages of three independent experiments. (d) All spectra 3 were subtracted fromspectrum 2 to obtain the fluorescence contribution of the TNP-ATP bound to the protein. FL, TraBFL; NT, TraBNT; CT, TraBCT; NT�CT,fluorescence expected from the sum of TraBNT and TraBCT. The arrow shows the decrease in fluorescence intensity between FL and NT�CT.(B) Measurement of binding equilibrium parameters. (a) Fluorescence-monitored titration of TNP-ATP binding to TraB proteins. Successivealiquots of TNP-ATP stock solutions were added to protein samples (1 �M) of TraBFL (E), TraBNT (�), or TraBCT (f) in buffer GFmb for TraBFLand TraBNT and in buffer GFsol for TraBCT, and the fluorescence intensity (excitation, 410 nm; emission, 545 nm) was recorded after each addition.Each plotted value represents the difference in fluorescence intensity between the TraB protein titration and the blank titration. The lines representthe best fit to the data generated using equation 3 in Materials and Methods: solid line, TraBFL; dashed line, TraBNT; dotted line, TraBCT. SeeMaterials and Methods for further details. (b) Displacement of bound TNP-ATP by ATP in TraB proteins. Successive aliquots of MgATP stocksolutions were added to a solution containing TraB proteins (1 �M) TraBFL (E), TraBNT (�), or TraBCT (f) and TNP-ATP (15 �M for TraBNTand TraBCT; 30 �M for TraBFL) in buffer GFmb for TraBFL and TraBNT and in buffer GFsol for TraBCT. The fluorescence intensity (excitation,410 nm; emission, 545 nm) was recorded after each addition. Each plotted value represents the difference in fluorescence intensity between theTraB protein titration and the blank titration. The lines represent the best fit to the data generated using equation 4 in Materials and Methodsand are as identified above. See Materials and Methods for further details.

TABLE 2. Summary of kinetic parameters of TraB, TraB fragments, and TraB mutants and comparison with TrwKa

Protein (NBD1 and/or NBD2)bKinetics with Michaelis-Menten equation Kinetics with Lineweaver-Burk plotc

Vmax (nmol min�1 mg�1) Vmax (mol min�1 mol�1) Km (mM) Vmax (nmol min�1 mg�1) Km (mM)

TraBFL (WT/WT) 126.2 � 4.6 12.9 � 0.4 0.59 � 0.07 120.5 0.52TraBFL (WT/KA) 25.0 � 0.8 2.6 � 0.1 0.60 � 0.09 23.9 0.54TraBFL (RA/WT) 30.1 � 0.5 3.1 � 0.1 0.31 � 0.02 30.03 0.30TraBNT (WT) 121.8 � 4.3 6.8 � 0.2 0.48 � 0.07 130 0.49TraBCT (WT) 45.1 � 1.5 2.2 � 0.1 0.62 � 0.07 42.2 0.52TrwK 48.4 0.7 NA NA

a Values were determined at 37°C.b See Table 1, footnote a, for an explanation of the protein designations.c NA, not available.

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TraBCT shows a Vmax of 2.20 � 0.07 mol of ATP hydrolyzedper min per mol of enzyme and a Km of 0.62 � 0.07 mM (Table2) (note that in all calculations, the monomer-equivalent con-centration was used). Subsequently, in order to assess the rel-evance of this ATPase activity, we constructed a Walker Aderivative of TraBCT carrying a point mutation K503A (seeMaterial and Methods), that has been shown to be of crucialimportance for ATPase activity in many proteins (22, 23). Theresulting mutant protein, TraBCT-KA, was purified in a similarway as the wild-type protein. DLS measurements indicatedthat this derivative forms hexamers of 353 kDa in GFacetate-sol

(Table 1) and is devoid of ATPase activity when tested inGFacetate-sol.

TraBFL purified from the soluble fraction in GFacetate-sol

(where it is hexameric [see above]) was also able to hydrolyzeATP. However, the kinetic parameters were significantlydifferent from those obtained for TraBCT (Fig. 5 and Table2). TraBFL shows a much higher Vmax than TraBCT, with avalue of 12.90 � 0.47 mol of ATP hydrolyzed per min permol of enzyme for TraBFL, but a similar Km of 0.59 � 0.07mM (Table 2).

Surprisingly, soluble hexameric TraBNT in GFacetate-sol wasalso able to hydrolyze ATP (Fig. 5). TraBNT shows a Vmax of

6.80 � 0.24 mol of ATP hydrolyzed per min per mol of enzyme,which is higher than that of TraBCT but lower than that ofTraBFL, and a similar Km of 0.48 � 0.07 mM compared to theother two proteins (Table 2). As mentioned previously, weidentified a poorly conserved ATP-binding site in the N-ter-minal domain of TraB (NBD1). In order to check the specificinvolvement of this NBD1, we designed a point mutation re-placing Arg53 by Ala (R53A) in TraBFL and in TraBNT (seeMaterials and Methods). The wild-type protein with the R53Amutation, TraBFL

RAWT, shows a marked decrease in itsATPase activity (3.10 � 0.24 mol of ATP hydrolyzed per minper mol of enzyme) compared to TraBFL (Table 2) whileTraBNT

RA no longer displays any measurable ATPase activity.These experiments confirm the specific involvement of theTraB NBD1 in the ATPase activity.

Interestingly, when assayed in GFsol, wild-type TraBCT,which is dimeric under such conditions (see above), is unableto hydrolyze ATP. This would indicate that only hexamericforms of TraB proteins exhibit ATPase activities. Consistentwith this observation, the membrane forms of TraBFL andTraBNT are unable to hydrolyze ATP in GFmb where the pro-teins are dimeric. The oligomeric state of these two proteins inGFacetate-mb was not investigated. However, neither TraBFL or

FIG. 4. Sequence alignment (Clustal W2) around the TraB NBD1 Walker A motif of VirB4 homologs. The shaded area shows the only threeVirB4 homologs that contain the NBD2 SecA-like motif.

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TraBNT extracted from the membrane exhibits ATPase activityin GFacetate-mb: given that detergent [0.01% Triton X-100(H)]does not affect the ATPase activity of hexameric TraBCT (thekinetic parameters are the same when TraBCT is tested forATP hydrolysis in the presence or absence of the detergent[data not shown]), lack of ATPase activity in membrane-ex-tracted TraBFL or TraBNT is not caused by detergent but,instead, may reflect the fact that these proteins remain dimericeven in the presence of acetate ions. This would suggest thatTraB in the membrane might be constitutively dimeric.

The effect of DNA binding on ATPase activity of TraBproteins was tested, and no difference in ATPase kinetic pa-rameters was observed. Thus, DNA binding does not affectATPase activity.

Conclusions. In the study presented here, we have unraveleda number of features of VirB4 proteins that were not apparentin TrwK, the other VirB4 protein for which extensive biochem-ical characterization has been carried out (2). TraB partitionsbetween the membrane and the cytoplasm, apparently adopt-ing two distinct oligomeric states, each of which is character-ized by a different ATP-hydrolyzing property. The form ex-tracted from the membrane is dimeric and unable to hydrolyzeATP although it is able to bind DNA and nucleotide. Inter-estingly, in a buffer with NaCl and no acetate ions, the solubleform of TraB is also dimeric and also unable to hydrolyze ATPwhile in the absence of NaCl but in acetate, it is hexameric andable to hydrolyze ATP. Thus, we conclude that TraB can hy-drolyze ATP only when in a hexameric state. TrwK differs fromTraB in being exclusively cytoplasmic and primarily mono-meric in low-salt buffer conditions. Higher oligomeric forms ofTrwK were observed in acetate buffer, but only up to 5% ofprotein was assessed to be hexameric, a form that was hypoth-esized to be the ATP-hydrolyzing form. The results with TraBare therefore less ambiguous in ascribing the ATP-hydrolyzingform of the protein to the hexameric form. Interestingly, TraBis able to transition from the soluble dimeric inactive form to

a soluble active hexameric form, demonstrating that the pro-tein is highly dynamic.

VirB4 proteins are likely to be located at the base of the coremachinery, embedded or associated with the inner membrane(16, 17). It is intriguing that TraB purifies as an inactive dimerwhen extracted from the membrane. It could be that in thephysiological conditions of the cell at the cytoplasmic face ofthe inner membrane (characterized by a low NaCl concentra-tion), the protein transitions to a hexamer even in the contextof the membrane. However, two other mechanisms could leadto an active TraB in the membrane: (i) a change in oligomer-ization from dimer to hexamer caused by the association withother T4S system components; (ii) the binding of an ATPase-activating protein, leaving TraB dimeric but inserting in transactivating residues missing at the ATP-binding interface in thedimer. Regarding the former, it is possible that the associationof VirB4 with the core complex might lead to a change in theoligomerization state. Possibly a VirB4-VirB11 interaction alsocould affect the oligomerization state of VirB4 as VirB11 isconstitutively hexameric. Regarding the latter, there are exam-ples of proteins that contain motifs known to be able to com-plete in trans ATPase active sites; one such motif is the well-characterized “Arg finger” of GAP proteins (1). VirB3 isknown to associate with VirB4 as both are sometimes found intandem in the same protein (4, 10). VirB3 has been proposedto locate VirB4 to the membrane for the VirB4 variants thatare not endowed with transmembrane segments. However,VirB3 could also influence VirB4 oligomerization and activityin such a way that, when embedded in the entire machinery, adimeric VirB4 could be rendered active.

Fragments of TraB recapitulate the behavior of the full-length protein in terms of oligomerization and ATP-hydrolyz-ing activity. Indeed, the soluble form of the C-terminal domainand both the soluble and membrane-bound forms of the N-terminal domain of TraB are dimeric and unable to hydrolyzeATP under NaCl-containing (GFsol or GFmb) buffer condi-

FIG. 5. Kinetic analysis of TraB and TraB domain ATPase activity. ATP hydrolysis activity was monitored by a coupled-enzyme assay (seeMaterials and Methods). (A) ATP hydrolysis rates are represented as a function of ATP concentration (x axis) and fitted using the Michaelis-Menten equation (represented by lines). The different data sets represent TraBFL (E and —), TraBNT (� and – –), TraBCT (f and ● ● ●),TraBFLRAWT (� and ● – – ●), and TraBFLWTKA (� and – ● ● ● –). All proteins were in GFacetate. The initial velocity rate (y axis) is expressed asmol of ATP hydrolyzed per min per mol of enzyme. (B) Lineweaver-Burk plot of the data presented in panel A. All the data sets have been fittedby linear regression.

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tions. Moreover, the soluble forms of these domains are able totransition from dimer to hexamer depending on the salt versusacetate conditions. However, remarkably—and demonstratinga property unique to TraB among VirB4 proteins—both do-mains are able to bind and hydrolyze ATP. This led us toidentify a novel cryptic but functional ATP-binding site in theN-terminal domain of the protein.

Another intriguing result is the ability of TraB and both ofits domains to bind DNA. A previous study of DNA-binding byVirB4-like proteins reported that these proteins were unableto bind DNA (29). Until now, only the coupling protein VirD4has been shown to bind DNA (32). The functional significanceof DNA binding by TraB is difficult to square with the reportedresult that VirB4 does not contact DNA directly during sub-strate transfer by the A. tumefaciens VirB/D4 T4S system (7).This could indicate that there are some differences betweenT4S systems and that, in the case of conjugation by thepKM101-encoded T4S system, DNA makes contact with TraB,perhaps relaying substrate transfer from the VirD4-homolog inthis system. However, in the absence of a mutation in TraBthat abrogates DNA binding, it would be difficult to speculatefurther as to whether DNA binding by this protein plays afundamental role in conjugation.

ACKNOWLEDGMENT

This work was funded by Welcome Trust grant 082227 to G.W.

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