Structural insights into the membrane-extracted dimeric form of the ATPase TraB from the Escherichia coli pKM101 conjugation system
© Durand et al; licensee BioMed Central Ltd. 2011
Received: 10 September 2010
Accepted: 25 January 2011
Published: 25 January 2011
Type IV secretion (T4S) systems are involved in secretion of virulence factors such as toxins or transforming molecules, or bacterial conjugation. T4S systems are composed of 12 proteins named VirB1-B11 and VirD4. Among them, three ATPases are involved in the assembly of the T4S system and/or provide energy for substrate transfer, VirB4, VirB11 and VirD4. The X-ray crystal structures of VirB11 and VirD4 have already been solved but VirB4 has proven to be reluctant to any structural investigation so far.
Here, we have used small-angle X-ray scattering to obtain the first structural models for the membrane-extracted, dimeric form of the TraB protein, the VirB4 homolog encoded by the E. coli pKM101 plasmid, and for the monomeric soluble form of the LvhB4 protein, the VirB4 homolog of the T4S system encoded by the Legionella pneumophila lvh operon. We have obtained the low resolution structures of the full-length TraB and of its N- and C-terminal halves. From these SAXS models, we derive the internal organisation of TraB. We also show that the two TraB N- and C-terminal domains are independently involved in the dimerisation of the full-length protein.
These models provide the first structural insights into the architecture of VirB4 proteins. In particular, our results highlight the modular arrangement and functional relevance of the dimeric-membrane-bound form of TraB.
Type IV secretion (T4S) systems are one of six secretion systems used to transport effector proteins or DNAs through the cell membrane of Gram-negative bacteria. These six secretions systems can be categorised into two classes. The first class of secretion systems mediates substrate transfer from the cytosol to the extracellular milieu in one step: substrates captured from the cytosol are released extracellularly without the need for a periplasmic intermediate . The second class encompasses a range of specialised outer membrane (OM) secretion systems: the substrate is first transported through the inner membrane (IM) to the periplasm via the general SecABYEG secretion machinery and then uses specialised OM systems for extracellular release [2, 3]. T4S systems belong to the first class.
T4S systems export proteins and DNA-protein complexes and fulfil a wide variety of functions, such as i- the conjugative transfer of plasmids and other mobile DNA elements to bacterial recipient cells, ii- the direct uptake of DNA from the extracellular milieu or iii- the delivery of protein or DNA substrates to eukaryotic target cells [4, 5]. T4S systems are used by several plant and human pathogens for virulence. Such bacterial pathogens include Agrobacterium tumefaciens, the causative agent of crown gall disease in plants, Bordetella pertussis, the agent responsible for whooping cough in children, and Helicobacter pylori, responsible for gastric ulcers and stomach cancer [6–9]. In addition, there are intracellular bacterial pathogens utilising T4S systems for virulence, such as Brucella suis, the causative agent of brucellosis, and Legionella pneumophila, the causative agent of Legionnaires' disease [10, 11].
T4S systems are generally composed of 12 protein components forming a macromolecular assembly inserted into the bacterial cell envelope . These proteins are named VirB1-VirB11 and VirD4, based on the widely used nomenclature of the model system, the A. tumefaciens VirB/D4 T4S system. Three ATPases are key components of the T4S system: VirD4, VirB11 and VirB4. VirB4 proteins are the largest and the most evolutionarily conserved proteins in T4S systems  but their function remains unclear. Although VirB4 proteins have clearly defined Walker A and Walker B motifs characteristic of ATPases , until very recently no ATPase activity had been demonstrated for any VirB4 homologues . However, two recent studies have shown that ATPase activities of VirB4 proteins are crucially dependent on solution conditions and on the oligomerisation state of VirB4 [14, 15]. For TrwK, the VirB4 homolog encoded by the R388 conjugative plasmid system, Rabel et al. initially reported that the protein exhibited no ATPase activity and was monomeric. However Arechaga et al. , in a subsequent study, reported an ATPase activity of TrwK in the presence of acetate ions, possibly due to a small proportion of an hexameric form of the protein. TraB, the VirB4 homolog encoded by the pKM101 conjugative plasmid system, also exhibits ATPase activity in the presence of acetate ions and is primarily hexameric under these solution conditions . Interestingly, TraB partitions between the cytosol and the inner membrane, and the membrane-extracted form does not exhibit ATPase activity, even in the presence of acetate ions . This membrane-extracted form of TraB was also shown to be dimeric. It was concluded that cytosolic TraB is in equilibrium between a dimeric form that binds DNA and nucleotides, but is unable to hydrolyze ATP, and an acetate-induced hexameric form able to hydrolyse ATP. TraB purified from the membrane is in the dimeric form, and is unable to transition to the hexameric form even in the presence of acetate ions . Interestingly, A. tumefaciens VirB4 was also shown to form active dimers in vivo, strongly supporting a functional role of this dimer, besides the hexameric form.
The structure of VirB4 proteins is still unknown, as they have resisted extensive crystallisation efforts either in the hexameric or the dimeric form. Attempts at visualising acetate-induced hexameric TraB by negative stained electron microscopy or small-angle X-ray scattering have failed . Recently, based on sequence similarities with TrwB (the VirD4 homolog from the plasmid R388 conjugation system), the A. tumefaciens VirB4 C-terminal domain was modelled, as an homo-hexameric ring  much like VirB11 and VirD4 . However no structural experimental data has yet backed this model, most probably because it has been impossible so far to stabilise and isolate the hexameric form of VirB4. Here we report the low resolution structure of the membrane-extracted dimeric form of TraB, using small-angle X-ray scattering (SAXS). We also performed a SAXS analysis of the N-terminal (TraBNT) and C-terminal (TraBCT) domains of TraB, and of the full-length monomeric LvhB4, the VirB4 homolog from the L. pneumophila T4S system, which represents the first in vitro study of a member of the L. pneumophila lvh T4S system. Altogether, our results provide the first insights into the architecture of the highly conserved VirB4 family of proteins.
Purification of TraB domains and LvhB4
For this study, TraBFL and TraBNT were both purified from the membrane fraction, while TraBCT, which is soluble and does not partition in the membrane, was purified from the soluble fraction. For comparison, we cloned, expressed and purified the full-length LvhB4, the VirB4 homolog from the L. pneumophila T4S system, for which no predicted TM domain was found. Indeed, LvhB4 purifies from the soluble fraction and not from the membrane fraction, demonstrating that the protein is not located in the membrane. All four proteins were purified to homogeneity using the same two-step purification strategy (Figure 1B and "Materials & Methods"). In SDS-PAGE the proteins migrate at their expected molecular mass: 102 kDa for TraBFL, 55 kDa for TraBNT, 49 kDa for TraBCT, and 94 kDa for LvhB4 (Figure 1B).
Size Determination of TraB domains and LvhB4
Theoretical and experimental Molecular Mass (MM) determination
146 - 242
66 - 146
Overall SAXS Parameters
Biophysical parameters estimated by SAXS
58.6 ± 0.6
198 ± 2
2.4 × 105
60.3 ± 0.7
195 ± 5
1.3 × 105
41.5 ± 0.5
150 ± 5
1.1 × 105
37.2 ± 0.6
120 ± 5
1.1 × 105
The radii of gyration measured on the different constructs are summarized in Table 2. Surprisingly, TraBFL and TraBNT have similar radii of gyration (58.6 ± 0.6 Å and 60.3 ± 0.7 Å respectively), in spite of the molecular mass of TraBNT being half of that of TraBFL. In contrast, the radii of gyration of TraBCT and LvhB4 are smaller (41.5 ± 0.5 Å and 37.2 ± 0.6 Å respectively). Proteins of similar radius of gyration may have very different shape and mass depending on their structure. Thus, TraBFL and TraBNT may have similar RG values but different structures. According to the SAXS results, TraBCT and consequently TraBFL are on average more compact than TraBNT. The RG of TraBNT reflects a less compact structure with a lower molecular mass, whereas, the RG of TraBFL results from a more compact structure and higher molecular mass, these two parameters counter-balancing each other to yield similar RG values.
We then calculated the pair-distance distribution function, P(r), from the SAXS curves (see "Materials & Methods"). The P(r) functions for all four constructs exhibited a bell-shaped curve with a slightly extended profile for the higher distances (data not shown), indicating a globular but somewhat elongated conformation. The comparison of the values obtained for the radius of gyration (RG) and for the maximum dimension (Dmax) for the four proteins gives an idea of their anisotropy. To obtain an estimation of the anisotropy of the protein, we calculated the ratio between RG and Dmax values for each construct. In the case of a sphere, this ratio is equal to 0.39, as the radius of a sphere is equal to (3/5)1/2R, where R is the radius of the sphere. Table 2 summarizes the values computed for all the constructs. The ratio RG/Dmax is 0.30 for TraBFL, 0.30 for TraBNT, 0.31 for TraBCT and 0.32 for LvhB4, significantly different than the value for a sphere, considering the error bars measured on the RG and Dmax. Interestingly, despite having different sizes, all four constructs exhibit the same anisotropy (ratio RG/Dmax of ~0.3), indicating that the proteins are rather anisotropic, and thus elongated.
Low Resolution Shapes from Ab Initio Modeling
Superposition and comparison of the TraB derived models
Comparison between TraBFL and LvhB4 and orientation of the TraB monomers
Comparison between TraBCT model and the homology-based structure of At-VirB4
The SAXS experiments reported here confirm that TraB and its N- and C-terminal domains are dimeric in the acetate-free solution conditions under which the experiments were conducted, indicating that both domains participate in the dimer interface. The structures of the two domains revealed elongated shapes, which in the full-length protein come together at a 45° angle. The superposition of the LvhB4 structure could resolve the ambiguity as to where the TraB monomer lies, and favored dimer 2 (Figure 5E), where the monomer would extend along the long axis of the dimer structure. Indeed, in the superimpositions of the two LvhB4 monomers onto the TraBFL dimer structure presented in Figure 6B and 6D, only the one aligning the LvhB4 monomers along the long axis of the TraB structure gives rise to an extended dimer interface. The configuration in Figure 5D (Dimer 1) would be expected to yield a less stable dimer than is observed. Also, a more extended conformation of TraB is consistent with our observation that TraB is susceptible to proteolysis and that limited proteolysis of TraB very rapidly yields TraBCT (data not shown).
VirB4 is a family of very conserved proteins that are essential components of T4S systems  However, recent biochemical studies have revealed that this family of proteins is more diverse than originally expected . For example, their oligomerisation state appears different depending on the system under investigation and the conditions under which they are studied. TraB has been shown to be in equilibrium between two oligomeric states, dimer and hexamer, dependent on the solution conditions, namely the presence or absence of acetate ion . TrwK appears to transition between a major monomeric form and minor hexameric form . The VirB4 homolog encoded by the cag pathogenicity island in H. pylori appears to be monomeric  and we show here that, under the solution conditions examined, the VirB4 homolog from L. pneumophila LvhB4 is monomeric. Hexamer formation appears to be required for ATP-hydrolyzing activity: indeed only hexameric forms of VirB4 homologs have been shown to exhibit ATPase activity [14, 15]. So far, only sparse information has been gathered about the function of the dimeric TraB. We recently reported its DNA and nucleotide binding activities , while A. tumefaciens VirB4 was shown to direct dimer formation when fused to the N-terminal portion of the cI repressor protein . The different subcellular localisation of TraB together with the recent characterization of a degenerated nucleotide binding site in its N-terminal domain  are features also observed in the SecA translocase, perhaps suggesting an evolutionary relationship between the two protein families [21, 22]. Finally, TraB in the context of the entire T4S machinery might interact with different partners. Indeed, its close association with TraA (the VirB3 homolog encoded by the pKM101 plasmid) or its documented protein-protein interactions with other T4S system components could induce the conformational changes necessary to reshape the active site or radically change its cell environment more specifically, in order to stabilise an active dimeric membrane-bound form.
The work presented here provides the first structural glimpse of a protein which is crucial to type IV secretion but has until now resisted X-ray crystallography or EM structural characterisation. It uncovers a modular structure that comes together in an extended dimer interface where the domains appear to "hug" each other. The dimers corresponding to each domain could easily be put together in the envelope of the full length protein and the structural model for LvhB4 helps suggest a potential model for the full-length protein. Intriguingly, the predicted TM segment locates within the N-terminal domain, not at the boundaries of the domain structure. This raises topological issues that can be resolved by a model invoking an orientation of the TraBNT domain facing the cytosolic side of the inner membrane, while the TraBCT domain would lie in the cytoplasm. This would be consistent with TraB being only superficially associated with the membrane, and therefore being able to partition between the membrane and the cytoplasm. It is also consistent with the dimeric structure proposed here. Docking of the TrwB protein (a potential structural homolog of the C-terminal domain of VirB4 proteins) within the envelope of the TraBCT provides further structural details. Finally, the dimeric model of TraB observed here suggests that there might be structural rearrangements required to fit the VirB4 dimeric structure into the 14-fold symmetrical core complex recently unravelled by the high resolution EM structure of the VirB7-VirB9-VirB10 complex  and confirmed by the subsequent crystal structure of its outer membrane-inserting part . Further studies will seek to elucidate the crystal structure of a VirB4 protein and also to visualize a complex of VirB4 bound to the core complex.
Cloning of TraB domains and LvhB4
Cloning of the full-length tra B gene (tra BFL, amino acids 1-866; Figure 1), the region encoding the N-terminal domain (tra BNT, amino acids 1-442; Figure 1) and the C-terminal domain (tra BCT, amino acids 448-848; Figure 1), together with the full-length lvh B4 gene (lvh B4 amino acids 1-826; Legionella pneumophila strain JR32) was as described in Durand et al.. All four constructs allow the expression of N-terminally His6-tagged recombinant proteins, referred to thereafter as TraBFL, TraBNT, TraBCT, or LvhB4. After DNA sequencing (MWG Biotech) to check that the sequences did not contained any mutation, the four plasmids were transformed by heat-shock in chemically competent BL21 star (DE3) cells (Invitrogen), for large scale production of the recombinant proteins.
Production and Purification of Recombinant Proteins
E. coli strain BL21 star/DE3 (Invitrogen) containing one of the recombinant plasmids was grown at 37°C in Terrific Broth supplemented with 100 μg/ml of Ampicillin (Sigma-Aldrich), until the culture reached an A600 nm of 1.2. Cultures were then shifted to 16°C for 1 h, before isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and growth continued for 15 h at 16°C. Cells were harvested by centrifugation, resuspended in 20 mM TrisHCl (pH7.5) and store at -20°C.
All subsequent steps were carried out at 4°C. TraBCT and LvhB4 were purified from cytoplasmic extracts as follow. The cells were defrosted and one tablet of Protease inhibitor cocktail EDTA free (Roche) was added, together with 300 mM NaCl and 1 mM β-mercapto-ethanol (βME). After cells were broken by two rounds through an EmulsiFlex-C5 homogeniser and DNA fragmentation by sonication, the lysate was clarified by centrifugation at 18,000 r.p.m. for 45 min in a Sorvall SS-34 rotor. The clarified lysate was loaded onto a HisTrapHP 5 ml column (GE Healthcare) equilibrated in buffer Asol (20 mM TrisHCl/pH7.5, 300 mM NaCl, 1 mM βME) plus 4% of buffer Bsol (20 mM TrisHCl/pH7.5, 300 mM NaCl, 1 mM βME, 500 mM Imidazole). The column was then washed with 100 ml of buffer Asol plus 8% buffer Bsol. Finally the proteins still bound to the column were eluted in a gradient from 4% to 100% of buffer Bsol in 100 ml. Eluted fractions containing either TraBCT or LvhB4 were pooled and concentrated in less than 4 ml before being loaded onto a HiPrep 16/60 Sephacryl S-300 HR column (Amersham) equilibrated in buffer GFsol (20 mM Tris/HCl pH 7.5, 50 mM NaCl, 1 mM βME). The proteins TraBCT and LvhB4 both eluted as a single peak. Fractions under this peak were pooled.
TraBFL and TraBNT were purified from membrane extracts as followed. The cells were defrosted and one tablet of protease inhibitor cocktail EDTA free (Roche) was added, together with 50 mM NaCl and 1 mM βME. After cells were broken by two rounds through an EmulsiFlex-C5 homogeniser and DNA fragmentation by sonication, unbroken cells were removed by centrifugation at 14,000 r.p.m. for 10 min in a Sorvall SS-34 rotor. Total membranes were pelleted by ultracentrifugation (45 min at 100,000 g, 4°C) and resuspended in buffer EB (20 mM Tris-HCl/pH 7.5, 50 mM NaCl, 1 mM βME, 1% (v/v) Triton® X-100) supplemented with one tablet of protease inhibitor cocktail EDTA free (Roche). Membrane-embedded proteins were extracted during 1 h at 4°C. The membrane extract was further clarified by ultracentrifugation (30 min at 100 000 g, 4°C). Triton® X-100 was only used for extraction, then it was replaced by the hydrogenated Triton® X-100(H) (Calbiochem) that does not absorb in UV. We further used a concentration of 0.01% Triton® X-100(H) (0.16 mM) since it was below the CMC of the detergent (0.2-0.9 mM), thus avoiding the formation of detergent micelles. The cleared extract was loaded onto a HisTrapHP 5 ml column (GE Healthcare) equilibrated in buffer Amb (20 mM TrisHCl/pH7.5, 300 mM NaCl, 1 mM βME, 0.01% Triton® X-100(H)) plus 4% of buffer Bmb (20 mM TrisHCl/pH7.5, 300 mM NaCl, 1 mM βME, 0.01% Triton® X-100(H), 500 mM Imidazole). The column was then washed with 100 ml of buffer Amb plus 8% buffer Bmb. Finally the proteins still bound to the column were eluted in a gradient from 4% to 100% of buffer Bmb in 100 ml. Eluted fractions containing either HisTraBFL or HisTraBNT were pooled and concentrated in less than 4 ml before being loaded onto a HiPrep 16/60 Sephacryl S-300 HR column (Amersham) equilibrated in buffer GFmb (20 mM Tris/HCl pH 7.5, 50 mM NaCl, 1 mM βME, 0.01% Triton® X-100(H)). The proteins TraBFL and TraBNT both eluted as a single peak. Fractions under this peak were pooled. Apparent molecular mass of proteins eluted from the gel filtration column was deduced from a calibration carried out with low and high molecular mass calibration kits (Amersham Biosciences). Determination of protein concentration was carried out by either using the theoretical absorption coefficients at 280 nm, which were obtained using the program ProtParam on the EXPASY server (available on the World Wide Web at http://www.expasy.ch/tools), or with the Bio-Rad protein assay reagent (Bio-Rad).
Dynamic Light Scattering (DLS)
Dynamic light scattering experiments were performed with a DynaPro-801 (Protein Solutions) at room temperature. All samples were filtered prior to the measurements (Millex syringe filters, 0.22 μm; Millipore Corp.). Diffusion coefficients were inferred from the analysis of the decay of the scattered intensity autocorrelation function. The hydrodynamic radius and the molecular mass (MM) of proteins in solution were both deduced from translational diffusion coefficients. All calculations were performed using the software provided by the manufacturer (Dynamics V5.25.44).
SAXS experiments were performed in two different locations. TraBFL and TraBNT were analysed on beamline X33  at EMBL-Hamburg on storage ring DORIS III of the Deutsches Elektronen Synchrotron (DESY) using a MAR 345 image plate detector. The scattering patterns from solutions of TraBFL at protein concentrations of 3, 5, 7.5, 10, and 13.5 mg/ml, and for TraBNT at protein concentrations of 1.3, 2.1, 4.9, and 8.2 mg/ml were measured in buffer GFmb. At a sample detector distance of 2.7 m and wavelength (λ) of 1.5 Å, the scattering vectors, q ranging from 0.0093 Å-1 to 0.50 Å-1 was covered (q = 4πsinθ/λ, where 2θ is the scattering angle). According to radiation damage tests, one frame of 2 min exposure time was recorded for every sample. The data were normalised to the intensity of the transmitted beam and radially averaged, and the scattering of the buffer was subtracted, as absolutely no trace of the presence of micelles was detected from the buffer scattering curve. The difference curves were scaled for protein concentration and extrapolated to yield the final composite scattering curves. Molecular mass calibration was made with BSA.
TraBCT and LvhB4 were analysed at the European Synchrotron Radiation Facility (Grenoble, France) on beamline ID02 as described previously . The scattering patterns from solutions of TraBCT at protein concentrations of 2.1, 3.7, 4.3, 6.1, and 8.2 mg/ml, and for LvhB4 at protein concentrations of 2, 2.9, 4.6, 6, 7.3, and 8.9 mg/ml were measured in buffer GFsol. The wavelength was 1.0 Å. The sample-to-detector distances were set at 1.0 m (TraBCT) and 1.5 m (LvhB4), resulting in scattering vectors, q ranging from 0.011Å-1 to 0.50 Å-1 and from 0.010Å-1 to 0.37 Å-1 respectively. All experiments were performed at 20°C. Absolute calibration was made with water.
SAXS Data Evaluation
All steps for data processing were performed using the program package PRIMUS . The experimental SAXS data for all samples were linear in a Guinier plot of the low q region, indicating that the proteins did not undergo aggregation. The radius of gyration R G was derived by the Guinier approximation I(q) = I(0) exp(-q 2 R G 2 /3) for qR G < 1.0. The radii of gyration R G , calculated for different protein concentrations, displayed a slight concentration dependence arising from particle interferences in solution. Interference-free SAXS profiles were estimated by extrapolating the measured scattering curves to infinite dilution. The molecular masses of the solutes were inferred from I(0) values, the forward scattering intensity, which is proportional to the molecular mass of the protein according to relationship MM ~I(0)/c, where c is the protein concentration. The intensity I(0) was experimentally inferred from the intercept of the linear fit in the Guinier plot Ln[I(q)] versus q 2 at low q values (qR G < 1.0). The program GNOM  was used to compute the pair-distance distribution functions, P(r). This approach also features the maximum dimension of the macromolecule, Dmax.
Ab Initio Modeling
The overall shapes of the entire assemblies were restored from the experimental data using the program GASBOR . The scattering profiles were fitted on the spectrum of each protein up to q = 0.37Å-1. GASBOR searches a chain-compatible spatial distribution of an exact number of dummy residues, centred on the Cα atoms of the protein amino acid residues. We used both symmetry operations P1 and P2 proposed by the program GASBOR. At least 10 low resolution models obtained from different runs were averaged using the program DAMAVER  to construct the average model representing the general structural features of each reconstruction.
This work has been funded by Welcome Trust grant 082227 to GW. We thank Prof. Dr. Hubert Hilbi, ETH Zürich Institute of Microbiology (Switzerland), for providing us with the genomic DNA of Legionella pneumophila. We acknowledge the European Synchrotron Radiation Facility and the Deutsches Elektronen-Synchrotron for provision of synchrotron radiation facilities and we would like to thank, Pierre Panine for assistance in using beamline ID02, and Manfred Roessle for assistance in using beamline X33.
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