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• W2912632708 abstract "TraB is an FtsK-like DNA translocase responsible for conjugative plasmid transfer in mycelial Streptomyces. Unlike other conjugative systems, which depend on a type IV secretion system, Streptomyces requires only TraB protein to transfer the plasmid as dsDNA. The γ-domain of this protein specifically binds to repeated 8-bp motifs on the plasmid sequence, following a mechanism that is reminiscent of the FtsK/SpoIIIE chromosome segregation system. In this work, we purified and characterized the enzymatic activity of TraB, revealing that it is a DNA-dependent ATPase that is highly stimulated by dsDNA substrates. Interestingly, we found that unlike the SpoIIIE protein, the γ-domain of TraB does not confer sequence-specific ATPase stimulation. We also found that TraB binds G-quadruplex DNA structures with higher affinity than TraB-recognition sequences (TRSs). An EM-based structural analysis revealed that TraB tends to assemble as large complexes comprising four TraB hexamers, which might be a prerequisite for DNA translocation across cell membranes. In summary, our findings shed light on the molecular mechanism used by the DNA-translocating motor TraB, which may be shared by other membrane-associated machineries involved in DNA binding and translocation. TraB is an FtsK-like DNA translocase responsible for conjugative plasmid transfer in mycelial Streptomyces. Unlike other conjugative systems, which depend on a type IV secretion system, Streptomyces requires only TraB protein to transfer the plasmid as dsDNA. The γ-domain of this protein specifically binds to repeated 8-bp motifs on the plasmid sequence, following a mechanism that is reminiscent of the FtsK/SpoIIIE chromosome segregation system. In this work, we purified and characterized the enzymatic activity of TraB, revealing that it is a DNA-dependent ATPase that is highly stimulated by dsDNA substrates. Interestingly, we found that unlike the SpoIIIE protein, the γ-domain of TraB does not confer sequence-specific ATPase stimulation. We also found that TraB binds G-quadruplex DNA structures with higher affinity than TraB-recognition sequences (TRSs). An EM-based structural analysis revealed that TraB tends to assemble as large complexes comprising four TraB hexamers, which might be a prerequisite for DNA translocation across cell membranes. In summary, our findings shed light on the molecular mechanism used by the DNA-translocating motor TraB, which may be shared by other membrane-associated machineries involved in DNA binding and translocation. Most membrane-associated motors involved in macromolecular transport across bacterial cell membranes belong to the superfamily of hexameric P-loop ATPases (1Iyer L.M. Makarova K.S. Koonin E.V. Aravind L. Comparative genomics of the FtsK-HerA superfamily of pumping ATPases: Implications for the origins of chromosome segregation, cell division and viral capsid packaging.Nucleic Acids Res. 2004; 32 (15466593): 5260-527910.1093/nar/gkh828Crossref PubMed Scopus (247) Google Scholar). These motors are able to couple the chemical energy provided by ATP hydrolysis to the transport of DNA and/or protein effectors through biological membranes. The FtsK/SpoIIIE family of translocases include proteins involved in the transfer of the bacterial chromosome between spatially separated compartments and are the most representative members of this family (2Demarre G. Galli E. Barre F.X. The FtsK Family of DNA Pumps.Adv. Exp. Med. Biol. 2013; 767 (23161015): 245-26210.1007/978-1-4614-5037-5_12Crossref PubMed Scopus (24) Google Scholar). FtsK and SpoIIIE proteins both play an important role during chromosomal segregation in bacteria, in cell division and sporulation processes, respectively. They are membrane-anchored proteins that have an N-terminal domain with several transmembrane helices (3Berezuk A.M. Goodyear M. Khursigara C.M. Site-directed fluorescence labeling reveals a revised N-terminal membrane topology and functional periplasmic residues in the Escherichia coli cell division protein FtsK.J. Biol. Chem. 2014; 289 (25002583): 23287-2330110.1074/jbc.M114.569624Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar); a motor domain, common to all RecA-like hexameric ATPases; and a C-terminal γ-domain that confers specificity in DNA binding and dictates the directionality of DNA transport. The γ-domain recognizes specific 8-bp DNA motifs, named KOPS for FtsK (FtsK orienting/polarizing sequence) and SRS (SpoIIIE recognition sequence) for SpoIIIE (4Bigot S. Saleh O.A. Cornet F. Allemand J.F. Barre F.X. Oriented loading of FtsK on KOPS.Nat. Struct. Mol. Biol. 2006; 13 (17041597): 1026-102810.1038/nsmb1159Crossref PubMed Scopus (72) Google Scholar, 5Ptacin J.L. Nollmann M. Becker E.C. Cozzarelli N.R. Pogliano K. Bustamante C. Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis.Nat. Struct. Mol. Biol. 2008; 15 (18391964): 485-49310.1038/nsmb.1412Crossref PubMed Scopus (87) Google Scholar). A close homolog of these proteins involved in the conjugative transfer of plasmids in Streptomyces was recently discovered (6Vogelmann J. Ammelburg M. Finger C. Guezguez J. Linke D. Flötenmeyer M. Stierhof Y.D. Wohlleben W. Muth G. Conjugal plasmid transfer in Streptomyces resembles bacterial chromosome segregation by FtsK/SpoIIIE.EMBO J. 2011; 30 (21505418): 2246-225410.1038/emboj.2011.121Crossref PubMed Scopus (58) Google Scholar). This FtsK-like homolog, TraB, mediates the transfer of the plasmid as dsDNA (7Possoz C. Ribard C. Gagnat J. Pernodet J.L. Guérineau M. The integrative element pSAM2 from Streptomyces: Kinetics and mode of conjugal transfer.Mol. Microbiol. 2001; 42 (11679075): 159-166Crossref PubMed Scopus (75) Google Scholar). The protein directs plasmid transfer by binding to a specific plasmid region named cis-acting locus of transfer (clt) (8Reuther J. Gekeler C. Tiffert Y. Wohlleben W. Muth G. Unique conjugation mechanism in mycelial streptomycetes: A DNA-binding ATPase translocates unprocessed plasmid DNA at the hyphal tip.Mol. Microbiol. 2006; 61 (16776656): 436-44610.1111/j.1365-2958.2006.05258.xCrossref PubMed Scopus (53) Google Scholar). The clt regions of different plasmids contain direct 8-bp repeats, termed TraB-recognition sequence (TRS), 3The abbreviations used are: TRSTraB-recognition sequenceT4SStype IV secretion system. which is recognized by the γ-domain of its corresponding TraB protein (6Vogelmann J. Ammelburg M. Finger C. Guezguez J. Linke D. Flötenmeyer M. Stierhof Y.D. Wohlleben W. Muth G. Conjugal plasmid transfer in Streptomyces resembles bacterial chromosome segregation by FtsK/SpoIIIE.EMBO J. 2011; 30 (21505418): 2246-225410.1038/emboj.2011.121Crossref PubMed Scopus (58) Google Scholar, 9Franco B. González-Cerón G. Servín-González L. Direct repeat sequences are essential for function of the cis-acting locus of transfer (clt) of Streptomyces phaeochromogenes plasmid pJV1.Plasmid. 2003; 50 (14597013): 242-24710.1016/S0147-619X(03)00063-5Crossref PubMed Scopus (14) Google Scholar). The predicted structure, domain organization, and DNA-binding characteristics of TraB suggest that the TraB conjugation system is derived from an FtsK-like ancestor (6Vogelmann J. Ammelburg M. Finger C. Guezguez J. Linke D. Flötenmeyer M. Stierhof Y.D. Wohlleben W. Muth G. Conjugal plasmid transfer in Streptomyces resembles bacterial chromosome segregation by FtsK/SpoIIIE.EMBO J. 2011; 30 (21505418): 2246-225410.1038/emboj.2011.121Crossref PubMed Scopus (58) Google Scholar). TraB-recognition sequence type IV secretion system. This conjugative mechanism is different from other plasmid-encoded conjugation complexes that translocate DNA through a type IV secretion system (T4SS). Conjugative T4SS consists of at least 12 proteins involved in the processing of the plasmid, the formation of a core channel complex, the assembly of a pilus for cell-to-cell contacts, and also in supplying energy for pilus biogenesis and substrate transport (10Cabezón E. Ripoll-Rozada J. Peña A. de la Cruz F. Arechaga I. Toward an integrated model of bacterial conjugation.FEMS Microbiol. Rev. 2015; 39 (25154632): 81-9510.1111/1574-6976.12085PubMed Google Scholar, 11Zechner E.L. Lang S. Schildbach J.F. Assembly and mechanisms of bacterial type IV secretion machines.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2012; 367 (22411979): 1073-108710.1098/rstb.2011.0207Crossref PubMed Scopus (132) Google Scholar). One of these proteins, known as coupling protein because it connects the DNA processing machinery to the secretion channel, is an ATPase that belongs to the FtsK-like family of proteins. The prototype for the T4SS coupling proteins is TrwB, from plasmid R388. TrwB is a hexameric DNA-dependent ATPase implicated in the transfer of the plasmid as an ssDNA molecule (12Tato I. Zunzunegui S. de la Cruz F. Cabezon E. TrwB, the coupling protein involved in DNA transport during bacterial conjugation, is a DNA-dependent ATPase.Proc. Natl. Acad. Sci. U.S.A. 2005; 102 (15919815): 8156-816110.1073/pnas.0503402102Crossref PubMed Scopus (89) Google Scholar). The diameter of the central pore in TrwB is 20 Å, which is large enough to accommodate ssDNA. In contrast, the FtsK pore is 30 Å wide, which allows for the passage of dsDNA (13Gomis-Rüth F.X. Moncalián G. Pérez-Luque R. González A. Cabezón E. de la Cruz F. Coll M. The bacterial conjugation protein TrwB resembles ring helicases and F1-ATPase.Nature. 2001; 409 (11214325): 637-64110.1038/35054586Crossref PubMed Scopus (282) Google Scholar, 14Massey T.H. Mercogliano C.P. Yates J. Sherratt D.J. Löwe J. Double-stranded DNA translocation: Structure and mechanism of hexameric FtsK.Mol. Cell. 2006; 23 (16916635): 457-46910.1016/j.molcel.2006.06.019Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Contrary to FtsK-like motors, TrwB-like proteins do not contain a γ-domain involved in the specific recognition of a plasmid sequence. Instead, substrate binding is mediated by the interaction with accessory proteins (15Tato I. Matilla I. Arechaga I. Zunzunegui S. de la Cruz F. Cabezon E. The ATPase activity of the DNA transporter TrwB is modulated by protein TrwA: Implications for a common assembly mechanism of DNA translocating motors.J. Biol. Chem. 2007; 282 (17599913): 25569-2557610.1074/jbc.M703464200Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and also by recognizing G-quadruplex structures on the DNA with high affinity (16Matilla I. Alfonso C. Rivas G. Bolt E.L. de la Cruz F. Cabezon E. The conjugative DNA translocase TrwB is a structure-specific DNA-binding protein.J. Biol. Chem. 2010; 285 (20375020): 17537-1754410.1074/jbc.M109.084137Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These secondary DNA structures have been proposed to act as loading sites for the motor. G-quadruplex structures are formed in G-rich DNA sequences by the pairing of four guanines in a planar array (17Williamson J.R. G-quartet structures in telomeric DNA.Annu. Rev. Biophys. Biomol. Struct. 1994; 23 (7919797): 703-73010.1146/annurev.bb.23.060194.003415Crossref PubMed Scopus (675) Google Scholar). These structural motifs are widespread in genomes and act as control elements in essential biological processes, such as replication or transcription (18London T.B. Barber L.J. Mosedale G. Kelly G.P. Balasubramanian S. Hickson I.D. Boulton S.J. Hiom K. FANCJ is a structure-specific DNA helicase associated with the maintenance of genomic G/C tracts.J. Biol. Chem. 2008; 283 (18978354): 36132-3613910.1074/jbc.M808152200Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Interestingly, KOPS, SRS, and TRS sequences are also G-rich sequences and, therefore, might form G-quadruplex structures that could act as loading sites. In this work, we have observed that TraB from plasmid pSVH1 binds G-quadruplex structures with higher affinity than TRS sequences. The γ-domain of TraB is not required for the recognition of these secondary structures. Therefore, the motor domain, common to TrwB-like proteins, is responsible for this DNA-binding mode. Comparative genomic analysis of the motor domain of FtsK-, SpoIIIE-, TraB-, and TrwB-like proteins revealed that these proteins are closely related, sharing an evolutionary common ancestor (19Cabezon E. Lanza V.F. Arechaga I. Membrane-associated nanomotors for macromolecular transport.Curr. Opin. Biotechnol. 2012; 23 (22189002): 537-54410.1016/j.copbio.2011.11.031Crossref PubMed Scopus (16) Google Scholar). It is tempting to speculate that they also share a similar mechanism of loading, recognizing secondary DNA structures to establish the first DNA contacts. We have also characterized the enzymatic activity of the protein, showing that TraB is a DNA-dependent ATPase, highly stimulated by dsDNA substrates. The structural analysis of the protein by EM showed the existence of high-order oligomeric complexes that might be formed by the assembly of four TraB hexamers. Although at present is not possible to know if the formation of these high-order oligomeric structures is relevant for TraB function in vivo, all together, these results shed light on the mechanism used by this family of membrane-associated motors for DNA binding and translocation. The soluble domain of TraB protein (TraBs), attached to a His-tag at the N terminus (Fig. 1A), was purified to homogeneity and analyzed for ATPase activity in the presence and absence of different DNA substrates under various conditions. The activity was very sensitive to pH, with an optimal range between 5.8 and 6.4 (Fig. 1B). In the absence of DNA, the activity of the protein was ∼2000 nmol ATP min−1 mg−1. ATP hydrolysis was stimulated 35-fold in the presence of dsDNA (Vmax = 72,950 ± 2360 nmol ATP min−1 mg−1), whereas ssDNA caused a 12-fold stimulation (Vmax = 24,870 ± 2380 nmol ATP min−1 mg−1) (Fig. 1C). These values were obtained with DNA substrates lacking the specific TRS recognition sequence. It is important to note that these values were much higher than previously reported data for a full-length strepII-TraB fusion protein (Vmax = 800 nmol ATP min−1 mg−1) (8Reuther J. Gekeler C. Tiffert Y. Wohlleben W. Muth G. Unique conjugation mechanism in mycelial streptomycetes: A DNA-binding ATPase translocates unprocessed plasmid DNA at the hyphal tip.Mol. Microbiol. 2006; 61 (16776656): 436-44610.1111/j.1365-2958.2006.05258.xCrossref PubMed Scopus (53) Google Scholar). Moreover, in that previous work the described ATPase activity was not DNA-dependent. The ATPase assays were performed at pH 8, which is not an optimal pH value for measuring this activity. As shown in Fig. 1B, TraBs activity shows a high dependence on low pH values. Because in the previous report a full-length strepII-TraB protein was used, we cannot exclude the possibility that the N-terminal domain had an effect on the DNA-dependent ATPase activity. More likely, the pH dependence observed here might explain the absence of DNA-dependent ATPase activity reported in those previous experiments. TraB has been reported to have a specific DNA-binding activity, recognizing 8-bp TRS motifs within the clt region of the pSVH1 plasmid (6Vogelmann J. Ammelburg M. Finger C. Guezguez J. Linke D. Flötenmeyer M. Stierhof Y.D. Wohlleben W. Muth G. Conjugal plasmid transfer in Streptomyces resembles bacterial chromosome segregation by FtsK/SpoIIIE.EMBO J. 2011; 30 (21505418): 2246-225410.1038/emboj.2011.121Crossref PubMed Scopus (58) Google Scholar). This mode of interaction with the DNA is reminiscent of the 8-bp KOPS and SRS motifs recognized by FtsK or SpoIIIE, respectively. In these proteins, the interaction with DNA motifs involves the γ-domain of the protein (5Ptacin J.L. Nollmann M. Becker E.C. Cozzarelli N.R. Pogliano K. Bustamante C. Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis.Nat. Struct. Mol. Biol. 2008; 15 (18391964): 485-49310.1038/nsmb.1412Crossref PubMed Scopus (87) Google Scholar, 20Löwe J. Ellonen A. Allen M.D. Atkinson C. Sherratt D.J. Grainge I. Molecular mechanism of sequence-directed DNA loading and translocation by FtsK.Mol. Cell. 2008; 31 (18722176): 498-50910.1016/j.molcel.2008.05.027Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In an attempt to determine whether the recognition of TRS motifs affected the activity of TraBs, ATP turnover was analyzed using complementary 45-mer oligonucleotides containing part of the clt sequence of the pSVH1 plasmid, which includes three copies of the 8-bp TRS sequence (Fig. 2A) (oligonucleotides C and D from Table 1). As shown in Fig. 2B (black bars), no significant differences were found in ATP turnover when compared with nonspecific complementary 45-mer oligonucleotides (oligonucleotides A and B from Table 1). Independently of the presence or absence of the specific clt sequence and TRS repeats, TraBs basal ATPase activity was always increased by 30-fold.Table 1Sequence of oligonucleotides used for substrate preparationOligoSequence (5′–3′)AAAGGACGAAAACCTGTGTAGTGTTATGCCACTACAATATTGCCGCBGCGGCAATATTGTAGTGGCATAACACTACACAGGTTTTCGTCCTTCaTRS sequences are underlined.GACCGGGCCCCGACCCGGACGCCGGAGTGCCCGACCCGGACGCCCDbG-clusters are indicated by bold letters.GGGCGTCCGGGTCGGGCACTCCGGCGTCCGGGTCGGGGCCCGGTCEaTRS sequences are underlined.ATGCCACTACAATATTGCCGCGACCCGGAGACCCGGAGACCCGGAFTCCGGGTCTCCGGGTCTCCGGGTCGCGGCAATATTGTAGTGGCATGATGCCACTACAATATTGCCGCTCCGGGTCTCCGGGTCTCCGGGTCHaTRS sequences are underlined.GACCCGGAGACCCGGAGACCCGGAGCGGCAATATTGTAGTGGCATIbG-clusters are indicated by bold letters.TCGCCACGTTTCGCCGTTTGCGGGGGTTTCTGCGAGGAACTTTGGa TRS sequences are underlined.b G-clusters are indicated by bold letters. Open table in a new tab Next, we analyzed the effect of the γ-domain on the ATPase activity of the protein. Previous data obtained with SpoIIIE protein indicated that the γ-domain was inhibiting the ATPase activity of the protein in the absence of DNA (21Besprozvannaya M. Pivorunas V.L. Feldman Z. Burton B.M. SpoIIIE protein achieves directional DNA translocation through allosteric regulation of ATPase activity by an accessory domain.J. Biol. Chem. 2013; 288 (23974211): 28962-2897410.1074/jbc.M113.484055Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Therefore, we purified TraBsΔγ (Fig. 1A and Fig. S1) and the activity of the two proteins was compared. Contrary to what was observed with SpoIIIE, TraBs and TraBsΔγ presented a similar ATP turnover (black and gray bars in Fig. 2B), both in the absence and in the presence of the two types of dsDNA substrates. Although the removal of the γ-domain did not affect the ATPase activity of the protein, previous studies had shown that the TraB γ-domain binds preferentially to TRS sequences over random DNA (6Vogelmann J. Ammelburg M. Finger C. Guezguez J. Linke D. Flötenmeyer M. Stierhof Y.D. Wohlleben W. Muth G. Conjugal plasmid transfer in Streptomyces resembles bacterial chromosome segregation by FtsK/SpoIIIE.EMBO J. 2011; 30 (21505418): 2246-225410.1038/emboj.2011.121Crossref PubMed Scopus (58) Google Scholar). Therefore, we performed electrophoretic mobility shift assays (EMSA) with the same 45-bp dsDNA fragments that were used in the ATPase assays, with and without the specific clt sequence. Fig. 2C shows that only the dsDNA fragment containing the specific sequence is retarded, with an apparent Kd value of 0.9 μm. Deletion of the γ-domain results in a DNA-binding defect, as previously reported (6Vogelmann J. Ammelburg M. Finger C. Guezguez J. Linke D. Flötenmeyer M. Stierhof Y.D. Wohlleben W. Muth G. Conjugal plasmid transfer in Streptomyces resembles bacterial chromosome segregation by FtsK/SpoIIIE.EMBO J. 2011; 30 (21505418): 2246-225410.1038/emboj.2011.121Crossref PubMed Scopus (58) Google Scholar). KOPS and SRS motifs are involved in directing motion during chromosome segregation of FtsK and SpoIIIE translocases, respectively (5Ptacin J.L. Nollmann M. Becker E.C. Cozzarelli N.R. Pogliano K. Bustamante C. Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis.Nat. Struct. Mol. Biol. 2008; 15 (18391964): 485-49310.1038/nsmb.1412Crossref PubMed Scopus (87) Google Scholar, 22Sivanathan V. Allen M.D. de Bekker C. Baker R. Arciszewska L.K. Freund S.M. Bycroft M. Löwe J. Sherratt D.J. The FtsK γ domain directs oriented DNA translocation by interacting with KOPS.Nat. Struct. Mol. Biol. 2006; 13 (17057717): 965-97210.1038/nsmb1158Crossref PubMed Scopus (83) Google Scholar). Both translocases move toward the terminus and, therefore, the motors can find these asymmetric motifs in two different orientations: permissive orientation (pointing to the terminus) and nonpermissive orientation (pointing to the origin). At present, there are different models that try to explain how the motors deal with these motifs (20Löwe J. Ellonen A. Allen M.D. Atkinson C. Sherratt D.J. Grainge I. Molecular mechanism of sequence-directed DNA loading and translocation by FtsK.Mol. Cell. 2008; 31 (18722176): 498-50910.1016/j.molcel.2008.05.027Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 23Graham J.E. Sherratt D.J. Szczelkun M.D. Sequence-specific assembly of FtsK hexamers establishes directional translocation on DNA.Proc. Natl. Acad. Sci. U.S.A. 2010; 107 (21048089): 20263-2026810.1073/pnas.1007518107Crossref PubMed Scopus (41) Google Scholar, 24Lee J.Y. Finkelstein I.J. Crozat E. Sherratt D.J. Greene E.C. Single-molecule imaging of DNA curtains reveals mechanisms of KOPS sequence targeting by the DNA translocase FtsK.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (22493241): 6531-653610.1073/pnas.1201613109Crossref PubMed Scopus (49) Google Scholar25Cattoni D.I. Chara O. Godefroy C. Margeat E. Trigueros S. Milhiet P.E. Nöllmann M. SpoIIIE mechanism of directional translocation involves target search coupled to sequence-dependent motor stimulation.EMBO Rep. 2013; 14 (23559069): 473-47910.1038/embor.2013.39Crossref PubMed Scopus (24) Google Scholar). A recent work on SpoIIIE has shown that permissive SRS sequences increase the ATPase activity more than 20-fold above the values obtained with random DNA or nonpermissive SRS (21Besprozvannaya M. Pivorunas V.L. Feldman Z. Burton B.M. SpoIIIE protein achieves directional DNA translocation through allosteric regulation of ATPase activity by an accessory domain.J. Biol. Chem. 2013; 288 (23974211): 28962-2897410.1074/jbc.M113.484055Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In an attempt to determine whether the stimulation of TraB ATPase activity could be linked to the sensing of the TRS motif in a particular orientation or to the existence of sufficient DNA for a correct loading, we analyzed the turnover of the protein in the presence of two types of synthetic dsDNA. These new dsDNA fragments were long enough to accommodate the assembly of the protein in any orientation and were designed in the same way as those used in the work with SpoIIIE (21Besprozvannaya M. Pivorunas V.L. Feldman Z. Burton B.M. SpoIIIE protein achieves directional DNA translocation through allosteric regulation of ATPase activity by an accessory domain.J. Biol. Chem. 2013; 288 (23974211): 28962-2897410.1074/jbc.M113.484055Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The substrates contained either three overlapping TRS sequences in a “permissive” orientation (3 × TRS) (oligonucleotides E and F from Table 1), or three overlapping TRS sequences in the reverse orientation (3 × revTRS) (oligonucleotides G and H from Table 1) (Fig. 3A). However, it is important to note that the termini “permissive” and “nonpermissive” are not related to the origin of the plasmid but to the two possible orientations that the sequences can acquire in the plasmid. As shown in Fig. 3B, the ATPase activity of the protein in the presence of the three overlapping TRS sequences, in any orientation, was similar to that obtained with random DNA. The values obtained for TraBs and TraBsΔγ proteins were very similar with all the dsDNA substrates tested. Therefore, we can conclude that the ATPase activity of TraBs is independent of the TRS sequence and its orientation. To analyze the binding preferences of TraB, we performed EMSA with the two types of substrates. The affinity was similar for both substrates (Kd values of 1.8 and 2.2 μm, respectively), but lower than that obtained for the substrate that contains three TRS sequences together with part of the clt sequence (Kd = 0.9 μm). This result might indicate that the affinity of TraBs for the clt sequence is not only determined by TRS motifs and it might need a larger DNA fragment. Streptomyces species and their plasmids have a high G + C content in their DNA. More specifically, the GC content of the clt region of plasmid pSVH1 containing the 8-bp TRS repeats is 80% (Fig. 2A). It is well-known that G-rich sequences are involved in the formation of G-quadruplex structures on DNA (G4 DNA) (17Williamson J.R. G-quartet structures in telomeric DNA.Annu. Rev. Biophys. Biomol. Struct. 1994; 23 (7919797): 703-73010.1146/annurev.bb.23.060194.003415Crossref PubMed Scopus (675) Google Scholar). Moreover, it has been shown that TrwB, a motor that belongs to the FtsK-like family which is involved in DNA transfer in Gram-negative bacteria, presents a high affinity for G-quadruplex structures (16Matilla I. Alfonso C. Rivas G. Bolt E.L. de la Cruz F. Cabezon E. The conjugative DNA translocase TrwB is a structure-specific DNA-binding protein.J. Biol. Chem. 2010; 285 (20375020): 17537-1754410.1074/jbc.M109.084137Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), suggesting that this type of secondary DNA structures might act as loading sites for this motor. These data, together with the fact that TraBs presents higher affinity for the clt sequence than for just overlapping TRS sequences, prompted us to analyze the interaction of TraBs with intermolecular G-quadruplex structures. To this end, G4 DNA was formed with the 45-mer oligonucleotide I (Table 1), following a specific protocol to obtain this type of DNA secondary structures, as reported previously (16Matilla I. Alfonso C. Rivas G. Bolt E.L. de la Cruz F. Cabezon E. The conjugative DNA translocase TrwB is a structure-specific DNA-binding protein.J. Biol. Chem. 2010; 285 (20375020): 17537-1754410.1074/jbc.M109.084137Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) (see also “Experimental procedures”). Gel shift assays indicated that TraBs binds G4 DNA with higher affinity than TRS sequences (Fig. 4A). Moreover, TraBsΔγ protein was able to bind G4 DNA almost to the same extent as TraBs, which indicates that the γ-domain is not required for the recognition of this type of structures on the DNA. The analysis and quantification of the TraBs gel shift assays with different DNA substrates showed an apparent Kd value of 23 nm for G4 DNA, 900 nm for dsDNA with the clt sequence, and 1.9 μm for dsDNA with three overlapping TRS sequences (Fig. 4B). As mentioned, TraBsΔγ protein was only able to bind G4 DNA, with a Kd value of 190 nm. All together, these results suggest that TraB might have an initial contact with the DNA by recognizing G-quadruplex structures, without the involvement of the γ-domain. The last purification step of TraBs, consisting of a size exclusion chromatography, showed two different oligomerization states of the protein (Fig. 5). The elution profile exhibited two major protein peaks: peak 1, corresponding to the void volume of the column, and peak 2, corresponding to an apparent molecular mass of 70 kDa (compatible with the predicted molecular mass of the monomer). The eluted fractions were analyzed by SDS-PAGE, confirming that both peaks corresponded to different forms of TraBs protein (data not shown). Variations on the pH value or on the glycerol content of the buffer shifted the balance in favor of a particular oligomeric state, suggesting there was an equilibrium between the two distinct oligomeric species (Fig. S2). EM analysis of fractions from peak 1 revealed a monodisperse sample of single particles, with an apparent diameter of ∼200 Å (Fig. 6A). A 3D reconstruction obtained after classification of these particles revealed a high-order structure, compatible with the assembly of four hexamers. A rotational symmetry analysis of the particles supported the existence of a 4-fold symmetry along the z axis.Figure 63D reconstruction of TraBs oligomer. A, TraBs from peak 1 was negatively stained with uranyl acetate and analyzed by EM. Particles were selected, aligned, and classified by maximum likelihood methods. A 3D reconstruction was obtained by projection matching, with imposed C4 symmetry in the final iterations. Scale bar represents 100 nm. B, fitting of TraBs in the EM maps. An atomic model of TraBs was generated by using atomic coordinates from FtsK of P. aeruginosa as template (2iuu.pdb and 2ve9.pdb coordinates for the motor domain and the γ-domain, respectively). The atomic coordinates were docked into the EM maps using SITUS package software.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To get a better estimation of the molecular mass of these high-order structures, samples were also loaded onto a Superose 6 column. Samples from peak 1 eluted with an estimated molecular mass of 1.15 MDa, which is compatible with an assembly of 24 TraBs monomers. Further, an hexameric molecular model of TraBs was generated by molecular threading using as template the atomic coordinates of the motor domain (PDB ID: 2IUU) (14Massey T.H. Mercogliano C.P. Yates J. Sherratt D.J. Löwe J. Double-stranded DNA tra" @default.
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