Research in Microbiology 153 (2002) 199–204
www.elsevier.com/locate/resmic
Mini-review
Structure and role of coupling proteins in conjugal DNA transfer
F. Xavier Gomis-Rüth a,∗ , Fernando de la Cruz b , Miquel Coll a,∗
a Institut de Biologia Molecular de Barcelona, C.S.I.C., c/Jordi Girona, 18-26, E-08034 Barcelona, Spain
b Departamento de Biología Molecular (Laboratorio asociado al C.I.B., C.S.I.C.), Universidad de Cantabria, c/Herrera Oria, s/n, E-39011 Santander, Spain
Received 15 August 2001; accepted 15 February 2002
First published online 14 March 2002
Abstract
Type IV secretory systems are transmembrane bacterial multiprotein complexes. They are pivotal for conjugation, bacterial-induced plant
tumour formation, toxin secretion and mammalian pathogen intracellular activity. These systems are involved in the spread of antibiotic
resistance genes among bacteria by enabling conjugative DNA transfer. When such translocons transport DNA, they require the assistance
of multimeric integral inner membrane proteins, the type IV coupling proteins. Its structural prototype is plasmid R388 TrwB protein,
responsible for coupling the relaxosome with the DNA transport apparatus during bacterial conjugation. Its monomeric molecular structure
is reminiscent of ring helicases and AAA ATPases. The quaternary structure is made up by six equivalent protomers featuring a flattened
sphere resembling F1 -ATPase, with a central channel traversing the particle, thus connecting cytoplasm and periplasm.  2002 Éditions
scientifiques et médicales Elsevier SAS. All rights reserved.
Keywords: Bacterial conjugation; Coupling protein; Type IV secretion systems; DNA transfer; Helicase; Three-dimensional structure; ATPase
1. Macromolecular secretion by bacteria
In order to organise their living strategies, bacteria often secrete macromolecules in to the environment to adapt
it to their own needs. For this purpose, they employ secretory systems traversing cell membranes that can be classified into five types (I to V) and that require proteins fuelled by nucleosyl triphosphate (NTP) hydrolysis [4,11,19,
28,29]. A distinct variant is type IV translocation, through
which (nucleo)proteins can be either secreted or directly injected into recipient cells. Type IV secretion is among the
five known macromolecular secretion systems, the only one
capable of DNA translocation [7]. It utilises a multiprotein
bacterial transmembrane organelle or translocon specialised
in the intercellular transfer of a variety of macromolecules.
The shuttled complexes may be nucleoprotein particles and
multicomponent protein associations that traverse the bacterial envelope into bacterial, plant and mammalian cells, i.e.,
even across kingdom boundaries, and into the extracellular
space [9,24]. The transmembrane organelle typically consists of 10–15 proteins, whose encoding genes are organ* Correspondence and reprints.
E-mail addresses: [email protected] (F.X. Gomis-Rüth),
[email protected] (M. Coll).
ised in a single gene cluster, making up two major structural
components, a filamentous surface appendage (pilus) and a
membrane-associated protein complex responsible for substrate translocation across the involved membranes [50].
2. Conjugation proceeds via type IV translocons
Bacterial conjugation is a contact-dependent process of
DNA transport often induced by extracellular or growthphase-dependent signals [27,44] and it employs such a
type IV pathway. It is enabled by conjugative plasmids or
transposons that control unidirectional DNA transfer from
a donor to a recipient cell, which then becomes a potential
donor. This results in adaptation of bacterial populations to
a changing environment, such as presence of antibiotics. It
is also a means to shuttle genes between different species
and even across kingdom barriers [18,48]. In Gram-negative
bacteria, two cell surface structures are required in the
donor cell: an extracellular filament (sex pilus) for initiating
physical contact between the donor and recipient cells and
a nucleoprotein conductance channel or conjugal pore for
the transmission of protein-bound DNA across the donor cell
envelope and into the recipient cell [10].
Macromolecular transfer during conjugation is regulated
by genes contained in the plasmid transfer region, further
0923-2508/02/$ – see front matter  2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
PII: S 0 9 2 3 - 2 5 0 8 ( 0 2 ) 0 1 3 1 3 - X
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F.X. Gomis-Rüth et al. / Research in Microbiology 153 (2002) 199–204
subdivisible into dtr and mpf [48]. Dtr proteins are involved
in DNA processing and in formation of a nucleoprotein complex known as relaxosome. Conjugative DNA processing
implies a strand-specific cleavage at the DNA origin of transfer (oriT) by dedicated nicking enzymes or relaxases. This
process is followed by a strand displacement reaction, which
generates a free single-stranded (ss) DNA transfer intermediate, the T-strand. The relaxosome then presumably moves
to the transport pore at the cytoplasmic side of the cell envelope. The DNA transport apparatus, a type IV secretion
system formed by 11–12 plasmid Mpf proteins [48], translocates the T-strand to the recipient cell. Conjugative plasmids
assist nonself-transmissible plasmids in DNA delivery to recipient cells and are capable of transkingdom DNA translocation [15]. Furthermore, these systems can transfer proteins
to recipient cells independently of DNA.
3. Type IV secretion and pathogenicity
Agrobacterium tumefaciens is a plant oncogenic pathogen
that induces phytohormone overproduction and crown gall
disease in plant cells [40]. It harbours a VirB type IV transporter that translocates a segment of a bacterial tumourinducing (Ti) plasmid DNA, the T-region, in the form of an
ss nucleoprotein T-complex, from the bacterium to the plant
cell. Transfer is regulated by a set of 20 virulence (Vir) proteins encoded by the Ti plasmid, 11 of which constitute the
transporter [44].
Other pathogens transfer toxic proteins. Bordetella pertussis, causing whooping cough in humans, has coopted a set
of conjugal transfer system genes, ptl [43], that are highly
similar to virB genes and that conduct the export of Pertussis toxin protein across the bacterial envelope and into
the medium. In Helicobacter pylori, the inductor of gastritis, peptic ulcer and gastric cancer, a pathogenicity island is
the main culprit of virulence. It codes for 31 Cag proteins, at
least 6 of which may contribute to a type IV system, which
translocates the CagA antigen into gastric epithelial cells.
There, the protein interferes with the normal host cell signalling.
Facultative intracellular pathogens are capable of survival
and multiplication within phagocytes [34]. These pathogens
possess a type IV secretion system postulated to secrete
macromolecules to the vacuolar membrane or to the cytoplasm of the host cell, which adjust the vacuolar environment to the bacterial requirements [9]. In particular, Legionnaire’s disease originating from Legionella pneumophila,
contains four icm/dot genes similar to A. tumefaciens virB
genes and to those of various conjugal transfer systems [16].
Brucella suis and Brucella abortus cause brucellosis in
many mammalian species, including humans, in the form
of chronic infections with bacteraemia [24]. Their genomes
also encode a secretion system consisting of 12 open reading
frames with homologues of the 11 VirB proteins of A. tumefaciens pTi. Finally, the recently reported genome se-
quence of typhus-causing Rickettsia prowazekii [2] has revealed seven homologues of A. tumefaciens VirB.
4. Coupling proteins enable type IV transport of DNA
Type IV transport systems require, apart from the apparatus directly responsible for macromolecular trafficking, a coupling protein (T4CP; [6]), when dedicated to
conjugation-like nucleoprotein transfer. All self-transmissible plasmids in Gram-negative bacteria contain such a homologue, as do many Gram-positive plasmids [48]. Coupling proteins may also be required for type IV-mediated
protein export in some cases, as reported for A. tumefaciens
and H. pylori [7,9,40]. Nonetheless, several type IV export
systems devoted to protein export only, like the Bordetella
Ptl system, lack T4CPs. T4CPs are integral inner membrane
proteins, with an N-terminal transmembrane moiety and a
C-terminal cytoplasmic part, that may interact with the cognate type IV transport machinery [20,22,25]. They possess
Walker A and B NTP binding motifs [42] and may be energised by NTP hydrolysis to couple or link transmissible
(nucleo)protein complexes and a macromolecular translocation apparatus [6,10,22,30]. Inactive T4CP mutants or association of the couplers with other proteins can lead to
conjugation-deficient phenotypes [10,22]. Studies on chimaeras made up by the T4CP of one species and the transport apparatus of another reveal that specificity resides on
the former [5] and that their C-terminal parts condition efficiency and specificity [31]. Some of these proteins can be
functionally exchanged against other members of the family (see below) [5]. The T4CP family comprises members
associated with nucleoprotein mobilisation like TrwB from
plasmid R388, TraD and TraG proteins from various Gramnegative plasmids, archetypal VirD4 from A. tumefaciens
pTi and related proteins.
5. The plasmid transfer region of pR388 and its
coupling protein
E. coli 33-kb plasmid R388 encodes resistance to sulphonamide and trimethoprim. Within its mpf region, 11 trw
genes (trwD-trwN) are similar to A. tumefaciens virB genes
[8,9,20,50]. The encoded proteins build up a functional
type IV secretion system (see Fig. 1a). The plasmid displays
the shortest dtr region known [20], just comprising oriT,
trwA, trwB, and trwC. TrwC is a relaxase/helicase and it is
required for both nick cleavage at oriT and T-strand unwinding [21] before transfer. TrwA is a small, tetrameric protein
that binds to two sites around oriT and enhances TrwC relaxase activity while repressing transcription of the trwABC
operon. These two proteins, TrwA and TrwC, together with
oriT DNA and the host-encoded integration host factor, form
the relaxosome in R388 (Fig. 1b; [23]). Plasmid R388 dtr region codes for a third protein, TrwB, a basic integral mem-
F.X. Gomis-Rüth et al. / Research in Microbiology 153 (2002) 199–204
201
Fig. 1. (a) Model showing a possible arrangement of the 11 mpf -encoded Trw proteins (TrwD-N) making up the type IV translocon engaged in T-strand
delivery from the donor cell to the recipient during conjugation of pR388 across both the inner and the outer membranes. It is based on the proposed locations
for the homologous VirB proteins [20,50]. Accordingly, both inner-membrane associated TrwD and inner membrane TrwK may be hexameric ring ATPases
putatively involved in transport activation, TrwL the exported pilin subunit involved in cell contact, TrwE a periplasmic protein of unknown function and TrwJ
and TrwM outer-membrane proteins of unknown function. Outer-membrane lipoprotein TrwH may be a nucleation centre, together with the covalently bound
outer membrane TrwF protein. TrwG is localised on the periplasmic face of the inner membrane, TrwI is a transmembrane candidate for pore formation and
TrwN an exported protein. (b) Scheme displaying the relaxosome of pR388 before oriT nicking, made up of 2 tetramers of TrwA, 2 molecules of integration
host factor and nicking enzyme/helicase TrwC, together with the dsDNA around oriT.
brane 507-residue protein [20,22] containing type A (motif I
or GXXGXGKS/T box) and type B (motif II or DExx box)
NTP-binding signatures described for helicases and the α
and β subunits of F1 -ATPase [42]. It is a T4CP responsible for coupling the relaxosome complex with the transport
pore [5,6]. Sequence analysis predicts the transmembrane
domain to be localised in the first 70 N-proximal residues.
A soluble fragment lacking these residues (TrwBN70)
binds a fluorescent ATP analogue [22], as expected from
the NTP binding signature. This binding is pivotal for conjugation, as a single point mutant affecting Walker site A
(K136T) leads to a completely transfer-deficient phenotype.
Other mutants affecting different catalytic residues at the active site are also transfer-deficient, implying that nucleotide
hydrolysis is necessary for protein function. TrwBN70
binds DNA nonspecifically, independently of NTP binding,
and this correlates with TrwC nick-cleavage enhancement.
Like the orthologs VirD4 and TraD, TrwB is anchored on the
cytoplasmic side of the inner membrane [25,26]. TraD has
also been shown to bind ss and double-stranded (ds) DNA
nonspecifically [26]. Orthologue TraG can replace TrwB in
the mobilisation of ColE1 and RSF1010 plasmids by R388,
but not in self transfer of R388 [5].
6. Structure of TrwB, a prototype for the T4CP family
The TrwBN70 protomer exhibits an elongated shape
reminiscent of an orange segment ([13]; Fig. 2a). It is composed of an all-α domain (AAD) facing the cytoplasm and
a nucleotide-binding domain (NBD) attached to the inner
membrane. The latter displays a α/β P-loop-containing
NTP hydrolase core and its architecture mainly comprises
a central twisted β-sheet (Fig. 2a), flanked by helices on
both sides. On the membrane-proximal side of this central
β-sheet, a small 3-stranded antiparallel sheet is inserted perpendicularly. Due to the absence of interactions with the (excised) transmembrane domain (TMD) preceding strand 1, its
constituting strands are only partially defined in the experimental TrwBN70 structure. On top of the NBD, at the
membrane distal part, the smaller AAD, comprising 7 helices, is inserted.
The quaternary structure is made up by 6 structurally
equivalent TrwBN70 protomers that intimately associate
to feature a spherical particle, slightly flattened at both poles
along its vertical axis, of overall dimensions 110 Å in diameter and 90 Å in height (Fig. 2b, c). A central channel
runs from the AAD-shaped cytosolic pole to the membraneproximal pole (formed by the NBDs), ending at the transmembrane pore formed by the (tentatively modelled) TMDs
(Fig. 2d).
This channel is restricted to a diameter of ∼7–8 Å at its
entrance at the cytoplasmic side. This is the narrowest point
of the channel that, at its membrane end, has an opening of
∼22 Å. At that point, a close contact is observed between
two strands of the small antiparallel sheet. This renders (if
we extrapolate these interactions to all molecules of the
hexamer) a 12-stranded cylinder of β-ribbons that may coat
the interior of the 12-helix transmembrane barrel formed
by the (predicted) helical segments of the 70-residue TMD
(Fig. 2d).
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F.X. Gomis-Rüth et al. / Research in Microbiology 153 (2002) 199–204
Fig. 2. (a) Ribbon diagram of a TrwBN70 protomer. The NBD and the perpendicularly attached 3-stranded β-sheet facing the membrane are depicted in dark
gray, the AAD in light grey. A sulphate anion shown as CPK spheres occupies the nucleotide-binding site. (b) Ribbon plot of the hexameric ring-like structure
of TrwBN70 displaying a RecA α/β NBD core domain [37] in a lateral view superimposed with its semi-transparent Connolly surface [13]. (c) Same as
(b) but axial view on the cytosolic surface. (d) Solid-surface representation of the complete TrwB particle, including the modeled transmembrane parts (grey
background), showing only 4 of the 6 protomers to allow a view into the central channel, putatively connecting cytosol with the periplasmic space across the
inner membrane.
7. Proteins with a similar fold
RecA is the structural prototype for RNA and DNA helicases and its homohexameric architecture was first described
by electron microscopy studies [46]. These proteins are
molecular motors involved in DNA metabolism fuelled by
NTP hydrolysis to perform nucleic-acid unwinding or strand
separation and translocation [37,41]. RecA is essential to genetic recombination and repair. Further extensive structural
studies have been undertaken with monomeric 3 -5 PcrA
DNA helicase [35,39]. TrwB reveals a stringent structural
similarity of its NBD with the equivalent part of RecA and
other RecA-like core encompassing enzymes, such as the
replicative helicase/primase of bacteriophage T7 [32], in addition to T7 gene 4 ring helicase [33] and PcrA DNA helicase [39].
AAA proteins (ATPases associated with a variety of
cellular activities) encompass an NBD with similar fold to
TrwB NBD, as reported for eukaryotes, prokaryotes and
archaebacteria. These proteins are specialised, chaperonelike enzymes that participate in membrane fusion and
trafficking, organelle biogenesis, proteolysis and protein
folding [38]. This family includes the δ subunit of the
clamp loader complex of E. coli DNA polymerase III [14],
N-ethylmaleimide-sensitive fusion protein (NSF) domain 2,
a cytosolic ATPase required for intracellular vesicle fusion
reactions [17], AAA ATPase p97, involved in homotypic
membrane fusion [49], and ATP-dependent protease HsIUHsIV [3].
Further striking structural similarity of TrwB is observed
with both α and β subunits of F1 -ATPase, part of the
membrane-associated F0 F1 -ATPase complex responsible for
energy conversion through axial rotation movement in mitochondria, chloroplasts, and bacteria [1], suggesting a similar function for TrwB (see below). Last not least, the recently solved six-clawed grapple-shaped structure of homohexameric H. pylori Cag525/HP0525 traffic ATPase, an
inner-membrane associated part of the bacterial type IV se-
F.X. Gomis-Rüth et al. / Research in Microbiology 153 (2002) 199–204
cretion system dedicated to pathogenic protein CagA export,
also reveals structural similarity in the NBD core [45].
However, when looking at the hexameric toroidal quaternary structures of the mentioned protein families, helicases, AAA ATPases and Cag525 appear more flat-topped
than TrwB, with weaker interaction between the constituting protomers. This weaker intermonomer interaction permits helicases to interchange aggregation stages and to form
helicoidal protein filament formation [35,47]. The compact
overall hexameric structure of TrwB (see Fig. 2b, c) due to
extensive interprotomer contacts, however, makes it difficult
to envisage other oligomerisation states. Its nearly spherical
shape (as well as the overall hexamer dimensions) is more
reminiscent of the F1 -ATPase α3 β3 heterohexamer, which
is a membrane-associated protein, as TrwB.
8. The NTP binding site of TrwB
The nucleotide binding site (NBS) of TrwB is located
at the interface between vicinal monomers and is less deep
and more accessible to bulk solvent than other NTP binding pockets of RecA-like family members, like F1 -ATPase
and PcrA [1,36,39]. It is shaped by two loops that feature the helicase superfamily motifs I (Walker box A) and
II (Walker box B or DExx box), also described for NTP
binding proteins in general [42]. On looking at the whole
particle, the NBSs are located ∼32 Å apart on superficial
cavities forming a belt around the hexamer in the middle
of the membrane-proximal half. Structures are available for
TrwBN70 in its nonliganded apo state, in complex with
nonhydrolysable NTP analogues, with a sulphate anion and
with ADP [12,13]. In contrast to helicases and F1 -ATPase [1,
33], in TrwB the six active sites can be considered as equivalent. When comparing these structures, no significant differences are observed in the protein scaffold upon NTP binding (apo vs. ATP-binding state), but significant conformational changes occur in the comparison between these models and the sulphate-bound structure, where the anion occupies the position of a left-behind γ phosphate group after ATP hydrolysis. These changes are transmitted from the
surface-located NBS to the interior channel, putatively playing a triggering role. A structure in complex with ADP in the
presence of Mg2+ cations (mimicking a left-behind nucleoside diphosphate after hydrolysis), however, does not display
these major changes.
Acknowledgements
We are most grateful to Gabriel Moncalián, Rosa PérezLuque, Robert Huber, Ana González and Isabel Usón for
assistance in the experimental parts of TrwB structure determination and to Matxalen Llosa for helpful discussions.
This study was supported by grants PB98-1631, BIO20001659 and 2FD97-0518 from the Ministerio de Educación y
203
Cultura and the Ministerio de Ciencia y Tecnología, Spain,
by grants 1999SGR188 and 2001SGR346 and the Centre
de Referència en Biotecnologia, both from the Generalitat de Catalunya, by the European Union (grants HPRI-CT1999-00017 and ERBFMGCECT980134 to EMBL Outstation Hamburg and grants HPRI-CT-1999-00022 and
ERBFMGECT980133 to EMBL Outstation Grenoble) and
by the ESRF (all to F.X.G.R and M.C.). The work of the F.C.
laboratory was supported by grant PB98-1106 from DGICYT, Ministerio de Ciencia y Tecnología, Spain.
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