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Structure of TrwB, a gatekeeper in bacterial conjugation
The International Journal of Biochemistry & Cell Biology 33 (2001) 839–843 www.elsevier.com/locate/ijbcb
Molecules in focus
Structure of TrwB, a gatekeeper in bacterial conjugation F. Xavier Gomis-Ru¨th, Miquel Coll * Institut de Biologia Molecular de Barcelona, C.S.I.C., c/Jordi Girona, 18 – 26, 08034 Barcelona, Spain Received 6 March 2001; accepted 10 April 2001
Abstract Bacterial conjugation implies a trans-membrane passage of DNA, mediated by proteins encoded in conjugative plasmids. This results in a spread of genetic information, including antibiotic resistance acquisition by pathogens. Special cases of conjugation are trans-kingdom gene transfer from bacteria to plants or fungi, and even bacterial sporulation and cell division. One of the main actors in this process is an integral inner membrane DNA-binding protein, called TrwB in the E. coli R388 conjugative system. It is responsible for coupling the single-strand DNA to be transferred from the donor to the acceptor cell in its complex with other proteins, with a type IV secretion system making up the mating apparatus. The TrwB protomer consists of two domains: a nucleotide-binding domain of a/b topology, similar to RecA and DNA ring helicases, and an all-a domain. The quaternary structure reveals an almost spherical homohexamer, strikingly similar to F1-ATPase. A central 20 A, wide channel traverses the hexamer, thus connecting cytoplasm with periplasm. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Bacterial conjugation; DNA transfer; Nucleotide-binding domain; Type IV secretion system; X-ray crystal structure
1. Introduction Conjugation is the major route for horizontal gene transfer between bacteria and a means for rapid import of new genetic material, including resistance acquisition against antibiotics by pathogens. It implies the transfer of one of the two DNA strands (T-strand) of a conjugative plasmid contained in the host to a recipient cell through a * Corresponding author. Tel.: + 34-93-4006149; fax: +3493-2045904. E-mail addresses: [email protected] (F.X. Gomis-Ru¨th), [email protected] (M. Coll).
type IV secretion system that connects donor and acceptor cells [1]. This process is controlled by plasmid-encoded proteins. One of these is TrwB, a basic (pI = 10) integral membrane protein of 507 residues [2] harbouring a nucleotide-binding site (NBS). It is encoded by Escherichia coli plasmid R388 and is responsible for coupling the T-strand nucleoprotein complex, the relaxosome, to the translocon. ATP binding is pivotal for conjugation, as a single-point mutant affecting the NBS Walker A motif (K136T) is completely transferdeficient [2]. Therefore, TrwB can be considered as a gatekeeper in the conjugative process.
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F.X. Gomis-Ru¨th, M. Coll / The International Journal of Biochemistry & Cell Biology 33 (2001) 839–843
TrwB-like proteins, responsible for recruiting the relaxosome or other macromolecules to be transferred and coupling them with the secretory system, have been described for a number of systems [1,3]. They can be grouped into a family of type IV secretion system coupling proteins (T4CPs). Among them is VirD4, a protein involved in bacteria-induced plant tumour development [4]. This family further encompasses the SpoIIIE/FtsK protein subfamily [2], whose members are essential in sporulation and cell division.
2. Structure The three-dimensional structure of a soluble fragment of TrwB lacking the N-terminally located 70-residue transmembrane part (TrwBDN70) unveils an orange slice (Fig. 1a) of approximate dimensions 90× 45 × 40 A, [5]. It can be subdivided into two domains: a cytosol-oriented all-a helical domain (all-a domain) and a membrane-proximal nucleotide binding a/b domain (nucleotide-binding domain). The latter is constituted by a central, twisted 9-stranded mixed b-pleated sheet (Fig. 1a), flanked by 4 and 7 helices on either side. On the cytosolic side of the nucleotide-binding domain, the smaller 7-helix alla domain is inserted between strand b4 and helix aL (Fig. 1a). Six TrwBDN70 protomers intimately associate to render a spherical particle, somewhat flattened at both poles, of overall dimensions 110 A, in diameter and 90 A, in height (Fig. 1b). A central channel traverses it connecting the cytosol with the periplasmic space across the inner membrane (Fig. 1c and d). The channel entrance is plugged by a crown of asparagine residues and restricted to a diameter of 8 A, . This is the narrowest part of the channel, which is 20 A, wide all along, and ends up at its membrane side with an opening
of 22 A, , although a narrower section may occur at the (tentatively modelled, see Fig. 1c and d) transmembrane domain (TD) in order to avoid membrane permeability. The function of this T4CP is regulated by the NBS, putatively involved in ATP hydrolysis. The NBSs are located at the C-terminal edges of the sheets on superficial cavities at interfaces between vicinal protomers, 32 A, apart. They are shaped by two segments containing the Walker A and the Walker B motifs, respectively, described for NTPbinding proteins [6]. Based on a strong resemblance with F1-ATPase [5,7], TrwB residues contributing to nucleotide anchoring can be suggested to be Thr132, Lys136 and Ser137. The proposed catalytic residues in F1-ATPase, however, do not match any residues in the present structure, suggesting a different enzymatic mechanism.
3. Synthesis Bacterial conjugation is regulated by plasmidencoded proteins of two gene regions in Gramnegative bacteria, the DNA transfer and replication proteins (Dtr) and the mating pair formation proteins (Mpf), that make up the type IV secretion system. E. coli R388 is a 33 kb enterobacterial conjugative plasmid that confers to its host sulfonamide and trimethoprim resistance. It encompasses the shortest dtr region known [8], coding only three proteins: TrwC displays relaxase and DNA helicase activities. TrwA is a transcriptional repressor. Together with the origin of transference (oriT DNA) and the hostencoded integration host factor, these two proteins constitute the relaxosome. The latter is attached to the translocon by means of the third protein encoded by the R388 dtr region, TrwB [3],
Fig. 1. (a) Diagram of a TrwBDN70 monomer. Strands are displayed as arrows and numbered (1 – 12), helices, as thick ribbons, are labelled with capital letters (A –R). The N- and C-termini, so as the constituting subdomains (all-a domain and nucleotide-binding domain) and the relative localisation of cytosol and periplasmic membrane are presented. (b) Ribbon representation of the complete TrwBDN70 homohexamer particle, as viewed from the cytosol in an axial view (left) and from a side view (right). The dimensions of the particle are indicated. (c) Ribbon plot of four TrwBDN70 monomers within a particle with the modelled transmembrane segments (residues 1 – 70). (d) Same as (c), but showing the corresponding Connolly surface to highlight the central channel that connects the cytosol (up) with the periplasm (down) across the inner membrane.
F.X. Gomis-Ru¨ th, M. Coll / The International Journal of Biochemistry & Cell Biology 33 (2001) 839–843
Fig. 1.
841
842
F.X. Gomis-Ru¨ th, M. Coll / The International Journal of Biochemistry & Cell Biology 33 (2001) 839–843
a T4CP. The R388 mpf region codes for 10 further proteins (TrwD-M), controlled by two promoters. These proteins make up the secretion system that physically connects donor and acceptor and through which the T-strand is proposed to pass.
4. Biological function T4CPs are responsible for coupling a macromolecular complex, including the DNA to be transferred from a donor to an acceptor cell, or molecules to be secreted to the extracellular space with the physical structures forming the pore, the secretory organelle. These proteins possess a nucleotide-binding domain, as found in other proteins, such as helicases. The latter use energy derived from the hydrolysis of ATP to separate double-stranded (ds) DNA or RNA into two single strands [9]. Those that act at the replication fork are ring-shaped hexameric enzymes that move along one strand of a DNA that is proposed to pass through the central cavity and catalyse the displacement of the complementary strand [10]. The strong structural resemblance of TrwB with DNA ring helicases suggests, by analogy, that the single DNA T-strand might pass through the central channel of the particle, as described for RuvB, that pumps dsDNA through the central channel of its hexameric ring [11] or as observed in the bacteriophage F29 connector particle during capsid assembly or infective dsDNA translocation [12,13]. This allows a working mechanism hypothesis to be formulated: ATP binding/ hydrolysis could induce a molecular switch mechanism affecting the channel or triggering a domain rearrangement (between the all-a domain and nucleotide-binding domain of each subunit) and therefore catalyse DNA binding and displacement through it (Fig. 2). A major hurdle against the ssDNA traversing the central channel is the closure at the cytoplasmic side, were it is as narrow as 8 A, . This occurs, however, only at the very initial section of the channel, being otherwise of the appropriate size ( 20 A, ) to accommodate a single DNA strand. According to this hypothesis, the present structure would represent
Fig. 2. Cartoon displaying the proposed working mechanism of TrwB and possibly other T4CPs engaged in ssDNA transfer. Putatively triggered by an interaction with relaxosome components (made up by TrwA, TrwC, and the integration host factor, IHF), the TrwB hexamer might undergo a conformational change at each all-a domain/nucleotide-binding domain interface resulting in an enlarged pore size, sufficient to thread the T-strand through the TrwB particle and to inject it into the periplasmic space.
a closed conformation, and the cytoplasmic gate would open upon activation, a movement that might be induced by interaction with relaxosome components.
5. Possible medical applications Over the past years, resistance of bacteria against antibiotics has grown to become a major sanitary problem, in both industrialised and developing countries. Formerly curable diseases, such as gonorrhea, typhoid, tuberculosis, whooping cough or sifilis, among many others, are rapidly becoming difficult to treat. Hospital-acquired infections, one of the major challenges in Europe and North America, result in 14 000 casualties just in the USA, mostly caused by resistant
F.X. Gomis-Ru¨ th, M. Coll / The International Journal of Biochemistry & Cell Biology 33 (2001) 839–843
bacterial strains. However, the battery of drugs, with which we can combat these pathogens, starts to become obsolete, as no new antibacterial class has been developed since 1970. In this context, new approaches may complement those strictly aimed to destroy the pathogen, such as trying to prevent the exchange of genetic information that confers resistance. As has been stated, T4CPs are crucial gatekeeper proteins, where a single point mutation can completely block the transference process. Thus, rational drug design strategies employing TrwB or its homologues as targets in order to prevent ATP binding/hydrolysis should open the spectrum of possibilities against antibiotic resistance spread. The reported atomic structure establishes the fundamentals for it.
[5]
[6]
[7]
[8]
[9] [10]
References [11]
[1] P.J. Christie, J.P. Vogel, Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells, Trends Microbiol. 8 (2000) 354 – 360. [2] G. Moncalia´ n, E. Cabezo´ n, I. Alkorta, M. Valle, F. Moro, J.M. Valpuesta, F.M. Gon˜ i, F. de la Cruz, Characterization of ATP and DNA binding activities of TrwB, the coupling protein essential in plasmid R388 conjugation, J. Biol. Chem. 274 (1999) 36117 – 36124. [3] E. Cabezo´ n, J.I. Sastre, F. de la Cruz, Genetic evidence of a coupling role for the TraG protein family in bacterial conjugation, Mol. Gen. Genet. 254 (1997) 400 –406. [4] A.C. Vergunst, B. Schrammeijer, A. den Dulk-Ras, C.M.T. de Vlaam, T.J.G. Regensburg-Tuı¨nk, P.J.J.
[12]
[13]
.
843
Hooykaas, VirB/D4-dependent protein translocation from Agrobacterium into plant cells, Science 290 (2000) 979 – 982. F.X. Gomis-Ru¨ th, G. Moncalia´ n, R. Pe´ rez-Luque, A. Gonza´ lez, E. Cabezo´ n, F. de la Cruz, M. Coll, The bacterial conjugation protein TrwB resembles ring helicases and F1-ATPase, Nature 409 (2001) 637 – 641. J.E. Walker, M. Saraste, M.J. Runswick, N.J. Gay, Distantly related sequences in the a- and b-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold, EMBO J. 1 (1982) 945 – 951. J.P. Abrahams, A.G.W. Leslie, R. Lutter, J.E. Walker, Structure at 2.8 A, resolution of F1-ATPase from bovine heart mitochondria, Nature 370 (1994) 621 – 628. M. Llosa, S. Bolland, F. de la Cruz, Genetic organization of the conjugal DNA processing region of the IncW plasmid R388, J. Mol. Biol. 235 (1994) 448 – 464. E.H. Egelman, Pumping DNA, Nature 409 (2001) 573 – 575. E.H. Egelman, X. Yu, R. Wild, M.M. Hingorani, S.M. Patel, Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases, Proc. Natl. Acad. Sci. USA 92 (1995) 3869 – 3873. C.A. Parsons, A. Stasiak, R.J. Bennett, S.C. West, Structure of a multisubunit complex that promotes DNA branch migration, Nature 374 (1995) 375 – 378. A.A. Simpson, Y. Tao, P.G. Leiman, M.O. Badasso, Y. He, P.J. Jardine, N.H. Olson, M.C. Morais, S. Grimes, D.L. Anderson, T.S. Baker, M.G. Rossmann, Structure of the bacteriophage F29 DNA packaging motor, Nature 408 (2000) 745 – 750. A. Guasch, J. Pous, B. Ibarra, F.X. Gomis-Ru¨ th, J.M. Valpuesta, N. Sousa, J.L. Carrascosa, M. Coll, The high resolution crystal structure of the bacteriophage F29 connector particle reveals the detailed architecture of a DNA translocation machine, submitted for publication.
Molecules in focus
Structure of TrwB, a gatekeeper in bacterial conjugation F. Xavier Gomis-Ru¨th, Miquel Coll * Institut de Biologia Molecular de Barcelona, C.S.I.C., c/Jordi Girona, 18 – 26, 08034 Barcelona, Spain Received 6 March 2001; accepted 10 April 2001
Abstract Bacterial conjugation implies a trans-membrane passage of DNA, mediated by proteins encoded in conjugative plasmids. This results in a spread of genetic information, including antibiotic resistance acquisition by pathogens. Special cases of conjugation are trans-kingdom gene transfer from bacteria to plants or fungi, and even bacterial sporulation and cell division. One of the main actors in this process is an integral inner membrane DNA-binding protein, called TrwB in the E. coli R388 conjugative system. It is responsible for coupling the single-strand DNA to be transferred from the donor to the acceptor cell in its complex with other proteins, with a type IV secretion system making up the mating apparatus. The TrwB protomer consists of two domains: a nucleotide-binding domain of a/b topology, similar to RecA and DNA ring helicases, and an all-a domain. The quaternary structure reveals an almost spherical homohexamer, strikingly similar to F1-ATPase. A central 20 A, wide channel traverses the hexamer, thus connecting cytoplasm with periplasm. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Bacterial conjugation; DNA transfer; Nucleotide-binding domain; Type IV secretion system; X-ray crystal structure
1. Introduction Conjugation is the major route for horizontal gene transfer between bacteria and a means for rapid import of new genetic material, including resistance acquisition against antibiotics by pathogens. It implies the transfer of one of the two DNA strands (T-strand) of a conjugative plasmid contained in the host to a recipient cell through a * Corresponding author. Tel.: + 34-93-4006149; fax: +3493-2045904. E-mail addresses: [email protected] (F.X. Gomis-Ru¨th), [email protected] (M. Coll).
type IV secretion system that connects donor and acceptor cells [1]. This process is controlled by plasmid-encoded proteins. One of these is TrwB, a basic (pI = 10) integral membrane protein of 507 residues [2] harbouring a nucleotide-binding site (NBS). It is encoded by Escherichia coli plasmid R388 and is responsible for coupling the T-strand nucleoprotein complex, the relaxosome, to the translocon. ATP binding is pivotal for conjugation, as a single-point mutant affecting the NBS Walker A motif (K136T) is completely transferdeficient [2]. Therefore, TrwB can be considered as a gatekeeper in the conjugative process.
1357-2725/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 1 ) 0 0 0 6 0 - 7
840
F.X. Gomis-Ru¨th, M. Coll / The International Journal of Biochemistry & Cell Biology 33 (2001) 839–843
TrwB-like proteins, responsible for recruiting the relaxosome or other macromolecules to be transferred and coupling them with the secretory system, have been described for a number of systems [1,3]. They can be grouped into a family of type IV secretion system coupling proteins (T4CPs). Among them is VirD4, a protein involved in bacteria-induced plant tumour development [4]. This family further encompasses the SpoIIIE/FtsK protein subfamily [2], whose members are essential in sporulation and cell division.
2. Structure The three-dimensional structure of a soluble fragment of TrwB lacking the N-terminally located 70-residue transmembrane part (TrwBDN70) unveils an orange slice (Fig. 1a) of approximate dimensions 90× 45 × 40 A, [5]. It can be subdivided into two domains: a cytosol-oriented all-a helical domain (all-a domain) and a membrane-proximal nucleotide binding a/b domain (nucleotide-binding domain). The latter is constituted by a central, twisted 9-stranded mixed b-pleated sheet (Fig. 1a), flanked by 4 and 7 helices on either side. On the cytosolic side of the nucleotide-binding domain, the smaller 7-helix alla domain is inserted between strand b4 and helix aL (Fig. 1a). Six TrwBDN70 protomers intimately associate to render a spherical particle, somewhat flattened at both poles, of overall dimensions 110 A, in diameter and 90 A, in height (Fig. 1b). A central channel traverses it connecting the cytosol with the periplasmic space across the inner membrane (Fig. 1c and d). The channel entrance is plugged by a crown of asparagine residues and restricted to a diameter of 8 A, . This is the narrowest part of the channel, which is 20 A, wide all along, and ends up at its membrane side with an opening
of 22 A, , although a narrower section may occur at the (tentatively modelled, see Fig. 1c and d) transmembrane domain (TD) in order to avoid membrane permeability. The function of this T4CP is regulated by the NBS, putatively involved in ATP hydrolysis. The NBSs are located at the C-terminal edges of the sheets on superficial cavities at interfaces between vicinal protomers, 32 A, apart. They are shaped by two segments containing the Walker A and the Walker B motifs, respectively, described for NTPbinding proteins [6]. Based on a strong resemblance with F1-ATPase [5,7], TrwB residues contributing to nucleotide anchoring can be suggested to be Thr132, Lys136 and Ser137. The proposed catalytic residues in F1-ATPase, however, do not match any residues in the present structure, suggesting a different enzymatic mechanism.
3. Synthesis Bacterial conjugation is regulated by plasmidencoded proteins of two gene regions in Gramnegative bacteria, the DNA transfer and replication proteins (Dtr) and the mating pair formation proteins (Mpf), that make up the type IV secretion system. E. coli R388 is a 33 kb enterobacterial conjugative plasmid that confers to its host sulfonamide and trimethoprim resistance. It encompasses the shortest dtr region known [8], coding only three proteins: TrwC displays relaxase and DNA helicase activities. TrwA is a transcriptional repressor. Together with the origin of transference (oriT DNA) and the hostencoded integration host factor, these two proteins constitute the relaxosome. The latter is attached to the translocon by means of the third protein encoded by the R388 dtr region, TrwB [3],
Fig. 1. (a) Diagram of a TrwBDN70 monomer. Strands are displayed as arrows and numbered (1 – 12), helices, as thick ribbons, are labelled with capital letters (A –R). The N- and C-termini, so as the constituting subdomains (all-a domain and nucleotide-binding domain) and the relative localisation of cytosol and periplasmic membrane are presented. (b) Ribbon representation of the complete TrwBDN70 homohexamer particle, as viewed from the cytosol in an axial view (left) and from a side view (right). The dimensions of the particle are indicated. (c) Ribbon plot of four TrwBDN70 monomers within a particle with the modelled transmembrane segments (residues 1 – 70). (d) Same as (c), but showing the corresponding Connolly surface to highlight the central channel that connects the cytosol (up) with the periplasm (down) across the inner membrane.
F.X. Gomis-Ru¨ th, M. Coll / The International Journal of Biochemistry & Cell Biology 33 (2001) 839–843
Fig. 1.
841
842
F.X. Gomis-Ru¨ th, M. Coll / The International Journal of Biochemistry & Cell Biology 33 (2001) 839–843
a T4CP. The R388 mpf region codes for 10 further proteins (TrwD-M), controlled by two promoters. These proteins make up the secretion system that physically connects donor and acceptor and through which the T-strand is proposed to pass.
4. Biological function T4CPs are responsible for coupling a macromolecular complex, including the DNA to be transferred from a donor to an acceptor cell, or molecules to be secreted to the extracellular space with the physical structures forming the pore, the secretory organelle. These proteins possess a nucleotide-binding domain, as found in other proteins, such as helicases. The latter use energy derived from the hydrolysis of ATP to separate double-stranded (ds) DNA or RNA into two single strands [9]. Those that act at the replication fork are ring-shaped hexameric enzymes that move along one strand of a DNA that is proposed to pass through the central cavity and catalyse the displacement of the complementary strand [10]. The strong structural resemblance of TrwB with DNA ring helicases suggests, by analogy, that the single DNA T-strand might pass through the central channel of the particle, as described for RuvB, that pumps dsDNA through the central channel of its hexameric ring [11] or as observed in the bacteriophage F29 connector particle during capsid assembly or infective dsDNA translocation [12,13]. This allows a working mechanism hypothesis to be formulated: ATP binding/ hydrolysis could induce a molecular switch mechanism affecting the channel or triggering a domain rearrangement (between the all-a domain and nucleotide-binding domain of each subunit) and therefore catalyse DNA binding and displacement through it (Fig. 2). A major hurdle against the ssDNA traversing the central channel is the closure at the cytoplasmic side, were it is as narrow as 8 A, . This occurs, however, only at the very initial section of the channel, being otherwise of the appropriate size ( 20 A, ) to accommodate a single DNA strand. According to this hypothesis, the present structure would represent
Fig. 2. Cartoon displaying the proposed working mechanism of TrwB and possibly other T4CPs engaged in ssDNA transfer. Putatively triggered by an interaction with relaxosome components (made up by TrwA, TrwC, and the integration host factor, IHF), the TrwB hexamer might undergo a conformational change at each all-a domain/nucleotide-binding domain interface resulting in an enlarged pore size, sufficient to thread the T-strand through the TrwB particle and to inject it into the periplasmic space.
a closed conformation, and the cytoplasmic gate would open upon activation, a movement that might be induced by interaction with relaxosome components.
5. Possible medical applications Over the past years, resistance of bacteria against antibiotics has grown to become a major sanitary problem, in both industrialised and developing countries. Formerly curable diseases, such as gonorrhea, typhoid, tuberculosis, whooping cough or sifilis, among many others, are rapidly becoming difficult to treat. Hospital-acquired infections, one of the major challenges in Europe and North America, result in 14 000 casualties just in the USA, mostly caused by resistant
F.X. Gomis-Ru¨ th, M. Coll / The International Journal of Biochemistry & Cell Biology 33 (2001) 839–843
bacterial strains. However, the battery of drugs, with which we can combat these pathogens, starts to become obsolete, as no new antibacterial class has been developed since 1970. In this context, new approaches may complement those strictly aimed to destroy the pathogen, such as trying to prevent the exchange of genetic information that confers resistance. As has been stated, T4CPs are crucial gatekeeper proteins, where a single point mutation can completely block the transference process. Thus, rational drug design strategies employing TrwB or its homologues as targets in order to prevent ATP binding/hydrolysis should open the spectrum of possibilities against antibiotic resistance spread. The reported atomic structure establishes the fundamentals for it.
[5]
[6]
[7]
[8]
[9] [10]
References [11]
[1] P.J. Christie, J.P. Vogel, Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells, Trends Microbiol. 8 (2000) 354 – 360. [2] G. Moncalia´ n, E. Cabezo´ n, I. Alkorta, M. Valle, F. Moro, J.M. Valpuesta, F.M. Gon˜ i, F. de la Cruz, Characterization of ATP and DNA binding activities of TrwB, the coupling protein essential in plasmid R388 conjugation, J. Biol. Chem. 274 (1999) 36117 – 36124. [3] E. Cabezo´ n, J.I. Sastre, F. de la Cruz, Genetic evidence of a coupling role for the TraG protein family in bacterial conjugation, Mol. Gen. Genet. 254 (1997) 400 –406. [4] A.C. Vergunst, B. Schrammeijer, A. den Dulk-Ras, C.M.T. de Vlaam, T.J.G. Regensburg-Tuı¨nk, P.J.J.
[12]
[13]
.
843
Hooykaas, VirB/D4-dependent protein translocation from Agrobacterium into plant cells, Science 290 (2000) 979 – 982. F.X. Gomis-Ru¨ th, G. Moncalia´ n, R. Pe´ rez-Luque, A. Gonza´ lez, E. Cabezo´ n, F. de la Cruz, M. Coll, The bacterial conjugation protein TrwB resembles ring helicases and F1-ATPase, Nature 409 (2001) 637 – 641. J.E. Walker, M. Saraste, M.J. Runswick, N.J. Gay, Distantly related sequences in the a- and b-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold, EMBO J. 1 (1982) 945 – 951. J.P. Abrahams, A.G.W. Leslie, R. Lutter, J.E. Walker, Structure at 2.8 A, resolution of F1-ATPase from bovine heart mitochondria, Nature 370 (1994) 621 – 628. M. Llosa, S. Bolland, F. de la Cruz, Genetic organization of the conjugal DNA processing region of the IncW plasmid R388, J. Mol. Biol. 235 (1994) 448 – 464. E.H. Egelman, Pumping DNA, Nature 409 (2001) 573 – 575. E.H. Egelman, X. Yu, R. Wild, M.M. Hingorani, S.M. Patel, Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases, Proc. Natl. Acad. Sci. USA 92 (1995) 3869 – 3873. C.A. Parsons, A. Stasiak, R.J. Bennett, S.C. West, Structure of a multisubunit complex that promotes DNA branch migration, Nature 374 (1995) 375 – 378. A.A. Simpson, Y. Tao, P.G. Leiman, M.O. Badasso, Y. He, P.J. Jardine, N.H. Olson, M.C. Morais, S. Grimes, D.L. Anderson, T.S. Baker, M.G. Rossmann, Structure of the bacteriophage F29 DNA packaging motor, Nature 408 (2000) 745 – 750. A. Guasch, J. Pous, B. Ibarra, F.X. Gomis-Ru¨ th, J.M. Valpuesta, N. Sousa, J.L. Carrascosa, M. Coll, The high resolution crystal structure of the bacteriophage F29 connector particle reveals the detailed architecture of a DNA translocation machine, submitted for publication.