Article
Structure of a VirD4 coupling protein bound to a
VirB type IV secretion machinery
Adam Redzej1,†, Marta Ukleja1,†, Sarah Connery1,†, Martina Trokter1, Catarina Felisberto-Rodrigues1,
Adam Cryar2, Konstantinos Thalassinos1,2, Richard D Hayward1,2, Elena V Orlova1,* &
Gabriel Waksman1,2,**
Abstract
Type IV secretion (T4S) systems are versatile bacterial secretion
systems mediating transport of protein and/or DNA. T4S systems
are generally composed of 11 VirB proteins and 1 VirD protein
(VirD4). The VirB1-11 proteins assemble to form a secretion machin-
ery and a pilus while the VirD4 protein is responsible for substrate
recruitment. The structure of VirD4 in isolation is known; however,
its structure bound to the VirB1-11 apparatus has not been deter-
mined. Here, we purify a T4S system with VirD4 bound, define the
biochemical requirements for complex formation and describe the
protein–protein interaction network in which VirD4 is involved. We
also solve the structure of this complex by negative stain electron
microscopy, demonstrating that two copies of VirD4 dimers locate
on both sides of the apparatus, in between the VirB4 ATPases. Given
the central role of VirD4 in type IV secretion, our study provides
mechanistic insights on a process that mediates the dangerous
spread of antibiotic resistance genes among bacterial populations.
Keywords bacterial conjugation; structure; type 4 secretion system; VirD4
Subject Categories Microbiology, Virology & Host Pathogen Interaction;
Structural Biology
DOI 10.15252/embj.201796629 | Received 30 January 2017 | Revised 9 August
2017 | Accepted 22 August 2017 | Published online 18 September 2017
The EMBO Journal (2017) 36: 3080–3095
Introduction
Type IV secretion (T4S) systems are membrane embedded multipro-
tein complexes, which are present, both in Gram-negative and
Gram-positive bacteria, as well as archaea (Wallden et al, 2010;
Trokter et al, 2014; Costa et al, 2015). They are used to transport a
variety of biomolecules across the bacterial envelope. Depending on
the type of transported substrates, they can be divided into three
groups (Alvarez-Martinez & Christie, 2009). The first group of T4S
systems are called conjugative T4S systems and members of this
group transfer DNA from a donor to a recipient cell (de la Cruz et al,
2010). This process contributes to the spread of antibiotic resistance
genes among different bacterial species and is also instrumental in
bacterial adaptation to environmental changes (Thomas & Nielsen,
2005). The second group of T4S systems are responsible for DNA
release or uptake to or from the extracellular milieu, respectively:
these T4S systems operate in bacterial species such as Neisseria
gonorrhoeae and Helicobacter pylori (Karnholz et al, 2006; Ramsey
et al, 2011). Finally, T4S systems can translocate effector molecules
into eukaryotic cells, which can result in many diseases such as
Legionnaires’ disease caused by Legionella pneumophila or brucel-
losis caused by Brucella spp (Llosa et al, 2009; Nagai & Kubori,
2011; Terradot & Waksman, 2011).
The conjugative T4S systems are generally composed of 12
proteins, named after the T4S system from Agrobacterium tumefa-
ciens responsible for transfer of the T-DNA of the Ti plasmid into
plants. In this system, T4S system components are termed VirB1-
VirB11 and VirD4 (Fronzes et al, 2009a). The central channel of the
T4S system, called the “core” complex, is formed from 14 copies of
the proteins VirB7, VirB9 and VirB10, and spans both membranes
(Fronzes et al, 2009b; Rivera-Calzada et al, 2013). The overall struc-
ture of the core complex appears as a two-layered ring with 14-fold
symmetry. The outer layer (O-layer) is made of full-length VirB7
and the C-terminal domains of VirB9 and VirB10 and is anchored in
the outer membrane via lipidated VirB7 and an a-helical barrel
(Chandran et al, 2009). The inner-layer (I-layer) is assembled from
the N-terminal parts of VirB9 and VirB10 with the very N-terminal
part of VirB10 extending into the cytosol and inserting a transmem-
brane helix into the inner membrane. Recently, the structure of a
larger complex containing eight VirB components including the
VirB3-10 proteins (a complex referred to thereafter as T4SS3-10) was
solved by negative stain electron microscopy (NS-EM), revealing the
structure of the inner membrane part of a T4S system or inner
membrane complex (IMC) composed of VirB3-6 and VirB8
(Low et al, 2014). The most prominent features of the IMC are two
1 Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck, London, UK
2 Division of Biosciences, Institute of Structural and Molecular Biology, University College of London, London, UK
*Corresponding author. Tel: +44 2076316833; E-mail: e.orlova@mail.cryst.bbk.ac.uk
**Corresponding author. Tel: +44 2076316845; E-mail: g.waksman@mail.cryst.bbk.ac.uk
†These authors contributed equally to this work
[The copyright line of this article was changed on 25 September 2017 after original online publication.]
The EMBO Journal Vol 36 | No 20 | 2017 ª 2017 The Authors. Published under the terms of the CC BY 4.0 license3080
barrel-like structures which are each divided into three layers: the
lower, middle and upper tiers. These barrels are connected by an
arch region to a stalk region, which links the IMC to the core
complex. While each of the barrels is comprised of six copies of
VirB4, the accurate location of VirB3, VirB5, VirB6 and VirB8 within
the IMC is not clear. Extending from the core complex outside of the
cell is an appendage called a pilus, which is a polymer of VirB2 and
is essential for making the connection between the recipient and
donor cells. Recent high-resolution cryo EM structure of the pilus
isolated from the F-like plasmid shows that the inside of the pilus is
covered with lipid head groups, which might facilitate the transfer of
the DNA between cells (Costa et al, 2016; Hospenthal et al, 2017).
The energy powering the T4S system is supplied by three
ATPases VirB4, VirB11 and VirD4, which are located on the cyto-
plasmic side of the T4S system. While VirB4 together with VirB11
are required for pilus biogenesis and substrate translocation, VirD4
plays the role of a coupling protein, responsible for the recruitment
of the substrate to the T4S system channel (Cabezon et al, 1997,
2015; Cascales & Christie, 2004). VirB11 has been reported as a
hexamer in different systems (Machon et al, 2002; Savvides et al,
2003; Hare et al, 2006). VirD4 and VirB4, though expected to form
hexamers, have been identified in monomeric and dimeric forms as
well (Gomis-Ruth et al, 2001; Schroder et al, 2002; Rabel et al,
2003; Arechaga et al, 2008; Mihajlovic et al, 2009; Durand et al,
2011; Pena et al, 2012; Wallden et al, 2012). All three ATPases
interact with one another and these interactions likely direct the two
processes in which T4S systems are involved: pilus biogenesis and
substrate secretion (Atmakuri et al, 2004; Ripoll-Rozada et al,
2013). VirD4 also interacts with the N-terminally located transmem-
brane helices of VirB10 (Llosa et al, 2003; Ripoll-Rozada et al, 2013;
Segura et al, 2013). Although the general organization of the
conjugative T4S system is well known, the details of the substrate
secretion mechanism are still unclear. Most of the knowledge about
the path of the secreted DNA comes from the A. tumefaciens system
where the proteins with which the DNA interacts on its way through
the secretion channel have been identified. During transfer, a single
strand of DNA makes successive contact with the VirD4 coupling
protein, and then the VirB11 ATPase, the inner membrane proteins
VirB6 and VirB8 and finally the core complex proteins VirB7 and
VirB9 (Cascales & Christie, 2004).
Conjugative transfer of plasmid DNA requires the processing of
the plasmid DNA by a large soluble complex containing 3–4
proteins, the largest of which is called the relaxase (Ilangovan et al,
2017). The relaxase is loaded onto a region of the plasmid called the
origin of transfer or oriT with the assistance of 2–3 auxiliary
proteins (Zechner et al, 2012; Ilangovan et al, 2015). Once loaded,
the relaxase nicks oriT at a site called nic, and covalently reacts to
the resulting 50-phosphate. It is this covalent relaxase–ssDNA
complex that is then recruited to the VirD4 coupling protein for
transport through the T4S system.
The overall goal of the study presented here is to investigate the
structure of the coupling protein VirD4 in the context of the entire
T4S system encoded by the R388 conjugative plasmid. We purify a
complex containing the eight VirB3-10 proteins (named TrwM-TrwE
in the R388 nomenclature) to which the VirD4 protein (TrwB) is
bound (a complex termed “T4SS3-10+D4”), determine the stoichio-
metry of TrwB/VirD4 within this complex, define the biochemical
requirements for complex formation by systematically deleting each
one of the T4S system IMC component proteins and evaluating their
impact on complex formation and characterize the protein–protein
interaction network of TrwB/VirD4 with T4S system components
within the machinery. Finally, we present the NS-EM structure of
the T4SS3-10+D4 complex, which identifies extra density correspond-
ing to TrwB/VirD4 within the T4S machinery, and confirm both
presence and location of TrwB/VirD4 using cross-linking mass spec-
trometry (XL-MS) and immuno-labelling followed by NS-EM. This
study reveals a remarkable IMC where each multimeric ATPase,
TrwK/VirB4 and TrwB/VirD4, is present in two copies and is located at
the nexus of the T4S system where they can couple protein and
DNA transport in a remarkably efficient manner.
Results and Discussion
Expression of functional T4S systems
R388 is a conjugative plasmid approximately 30 kb in size, which
constitutively expresses the T4S system and relaxosome genes
(Bolland et al, 1990; Llosa et al, 1994). In this conjugative system,
the virB1-11 genes are called trwN-D while the genes encoding the
VirD4 coupling protein and the relaxase are called trwB and trwC,
respectively. Relaxosome formation requires two auxiliary proteins,
plasmid-encoded TrwA, and host-encoded IHF. trwN-D and trwABC
are located in two operons, the expression of which is coordinated
by three distinct promoters (de Paz et al, 2005; Fernandez-Lopez
et al, 2006). For clarity, we will use both the “Trw” and “VirB”
nomenclatures.
For the purpose of this study, three requirements for cloning
were considered key: (i) sufficient material for NS-EM analysis
should be obtained, (ii) tags should be easily engineered within the
vectors employed, and (iii) all combinations of constructs should
drive the production of functional T4S systems. All constructs gener-
ated in this study are listed in Appendix Table S1.
First, the trwN/virB1-trwD/virB11 operon was cloned into the vector
pBADM11 (referred to as pBADM11_trwN/virB1-trwD/virB11), which is
under the regulation of the inducible ara promoter. In parallel, the
trwABC genes encoding for the relaxosome components as well as
the coupling protein were cloned into the pCDFduet vector (referred
to as pCDF_trwABC). A third construct was generated containing
the R388 plasmid oriT sequence in the pRSF plasmid (referred to as
pRSF_oriT). All plasmids are compatible, having a different origin of
replication. As it is crucial to ascertain that expressed T4S system
and relaxosome components are assembled properly, bacteria
containing the combination of three plasmids (the pBADM11_
trwN/virB1-trwD/virB11 under arabinose control, the trwABC-containing
plasmid under IPTG control and the oriT-containing plasmid) were
tested for their ability to conjugate the oriT –containing plasmid into
recipient cells. Conjugation experiments were conducted, and conju-
gation efficiencies were determined as described in Materials and
Methods. As reported in Table 1, the three-plasmid system efficiently
mediates conjugative transfer. These results are also consistent with
the findings by Bolland et al (1990) that the trw genes can be taken
out of their R388 plasmid context to mediate conjugation.
For purification of T4S system complexes, the pBADM11_
trwN/virB1-trwD/virB11 expression clone was modified. Previous
studies on T4S system have exploited a Strep-tag located at the
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C-terminus of VirB10 (Chandran et al, 2009; Fronzes et al, 2009b;
Wallden et al, 2012; Rivera-Calzada et al, 2013; Low et al, 2014).
Thus, a sequence encoding a Strep-tag was introduced at this
position of the trwE/virB10 gene. The resulting clone is named
pBADM11_trwN/virB1-trwE/virB10Strep_trwD/virB11.
Using this construct, two additional clones were produced: i—
pBADM11_trwN/virB1-trwE/virB10Strep_trwD/virB11_HistrwB/virD4, with
the TrwB/VirD4-encoding gene located after the sequence encoding
TrwD/VirB11 and N-terminally His-tagged, and ii—pBADM11_
trwB/virD4His_trwN/virB1-trwE/virB10Strep_trwD/virB11, with the TrwB/VirD4-
encoding gene inserted before the sequence encoding TrwN/VirB1 and
C-terminally His-tagged. To test whether these constructs induce
formation of functional T4S systems, we generated a variant of
pCDF_trwABC where the trwB/virD4 gene was deleted (resulting in a
construct named pCDF_trwAC). The pBADM11_trwN/virB1-trwE/virB10Strep_
trwD/virB11_HistrwB/virD4 or pBADM11_trwB/virD4His_trwN/virB1-
trwE/virB10Strep_trwD/virB11 constructs were then assayed using
pCDF_trwAC and pRSF_oriT for their ability to mediate conjugation
of pRSF_oriT. As shown in Table 1, conjugation is still observed but
reduced by approximately 10-fold in both cases, indicating that these
constructs still assemble functional T4S system and relaxosome
complexes, albeit at a reduced efficiency compared to wild type.
Conjugation does not occur when trwB/virD4 is removed.
VirD4 binds to the T4SS3-10 complex
Having obtained functional conjugative T4S machineries, we next
asked whether any of these machineries could be extracted from the
bacterial envelope and purified. We therefore induced expression
of the pBADM11_trwN/virB1-trwE/virB10Strep_trwD/virB11_HistrwB/virD4
or the pBADM11_trwB/virD4His_trwN/virB1-trwE/virB10Strep_trwD/virB11
constructs and submitted the detergent-solubilized membrane
extracts to two consecutive tag-affinity chromatography steps, first
using the Strep-tag on TrwE/VirB10 and then the His tag on
TrwB/VirD4. The resulting materials were analysed by SDS–PAGE.
Both constructs yielded similar results, and therefore, all subsequent
experiments were carried out using the pBADM11_trwN/virB1-
trwE/virB10Strep_trwD/virB11_HistrwB/virD4 construct. We observed 10
bands (Fig 1A, left panel). These were identified by mass spectrom-
etry to be (from higher to lower molecular weight) TrwK/VirB4,
TrwB/VirD4, TrwE/VirB10, TrwG-F/VirB8-9, TrwI/VirB6, TrwJ/VirB5, TrwM/VirB3
and TrwH/VirB7. The presence of TrwB/VirD4 was confirmed by puri-
fying the T4SS3-10 complex from cells expressing the pBADM11_
trwN/virB1-trwE/virB10Strep_trwD/virB11 construct. In the SDS–PAGE pro-
file of the complex purified from TrwB/VirD4-producing cells, a clear
band of a size between 50 and 60 kDa is observed, which is absent
in the T4SS3-10 complex (Fig 1A, right panel). The theoretical molec-
ular weight of TrwB/VirD4 incorporating 10 additional Histidine resi-
dues is 57 kDa. The Western blot analysis using TrwB/VirD4
antibodies (this study; see Materials and Methods for TrwB/VirD4
antibody production) confirmed that this band corresponds to the
TrwB/VirD4 protein (Fig EV1A). All other bands observed in the
SDS–PAGE analysis of the complex purified from TrwB/VirD4-producing
cells are also present in the T4SS3-10 complex. This includes OmpC
and OmpA, contaminants previously proven to be minor (Low
et al, 2014). Thus, the complex purified from TrwB/VirD4-producing
cells is the T4SS3-10 complex to which VirD4 is bound. We term
this complex the T4SS3-10+D4 complex.
We next determined the stoichiometry of TrwB/VirD4 within the
T4SS3-10+D4 complex using cysteine labelling (Fig 1B). Free thiol
groups within the proteins of the complex were reacted using Alexa
Fluor633 coupled to maleimide. The labelled sample of the complex
was analysed using SDS–PAGE, and the fluorescence intensity of
the bands was quantified. The TrwE/VirB10 protein as a part of the
TrwH/VirB7-TrwF/VirB9-TrwE/VirB10 core complex alone or of the
T4SS3-10 complex is known to be present in 14 copies (Fronzes et al,
2009b; Low et al, 2014). Therefore, the fluorescence signal quanti-
fied from the TrwE/VirB10 band was used as a reference to determine
the stoichiometry of the other components. As a result, the amount
of TrwB/VirD4 within the sample can be derived as 4.5  0.2
TrwB/VirD4 subunits per complex (calculated from three independent
preparations of the T4SS3-10+D4 complex; Fig 1B). All other compo-
nents in the T4SS3-10+D4 complex were present in the same stoi-
chiometry observed previously in the T4SS3-10 complex.
Systematic deletions of single T4SS IMC components reveal
different subcomplexes of the T4SS3-10+D4 complex
To better understand how the absence of different components of
the T4SS affect T4SS3-10+D4 complex formation, single trw/virB gene
deletion mutants within the pBADM11_trwN/virB1-trwE/virB10Strep_
trwD/virB11_HistrwB/virD4 construct were generated. These constructs
will be referred to as DtrwX/virBY deletion constructs, X (a letter)
or Y (a number) identifying the component gene deleted from the
cluster using the Trw or VirB nomenclature, respectively
(Appendix Table S1). Only deletions of IMC components (TrwL/VirB2,
TrwM/VirB3, TrwK/VirB4, TrwJ/VirB5, TrwI/VirB6, TrwG/VirB8 and
TrwD/VirB11) were investigated as core OMC components,
TrwH/VirB7, TrwF/VirB9 and TrwE/VirB10, are already known to be
indispensable for complex assembly (Fronzes et al, 2009b), and
TrwN/VirB1 is not an essential part of the T4S system (Berger &
Christie, 1994). These constructs were then used to produce and
purify subcomplexes using the same purification protocol employed
for the purification of the T4SS3-10+D4 complex, that is a pull-down
using the Strep-tag on TrwE/VirB10 followed by a pull-down using
the His tag on TrwB/VirD4. Resulting complexes were analysed using
SDS–PAGE to compare which proteins are still present within the
complex in the absence of individual T4S system component
(Fig 1C). Three distinct complexes could be observed. In the
absence of the TrwL/VirB2, TrwJ/VirB5 or TrwD/VirB11, all of the other
components of the T4SS3-10+D4 complex are still present. Therefore,
none of the pilus subunits or TrwD/VirB11 are essential for the stabil-
ity of the T4SS3-10+D4 complex. However, deletion of TrwM/VirB3 or
Table 1. Conjugation efficiencies of various R388 T4SS constructs.
T4S plasmid
Relaxosome
plasmid
Mating
efficiency
pBADM11_trwN/virB1-trwD/virB11 pCDF_trwABC 0.5
pBADM11_trwN/virB1-trwD/virB11 pCDF_trwAC < 10
6
pBADM11_trwN/virB1-trwE/virB10Strep_
trwD/virB11_HistrwB/virD4
pCDF_trwAC 0.07
pBADM11_ trwB/virD4His_trwN/virB1-
trwE/virB10Strep_trwD/virB11
pCDF_trwAC 0.05
The mating efficiencies obtained with plasmids expressing different
variations of the T4S system genes from R388 are reported.
The EMBO Journal Vol 36 | No 20 | 2017 ª 2017 The Authors
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A B
C
Figure 1. SDS–PAGE analysis of purified T4SS3-10+D4 complex, comparison with the T4SS3-10 complex, stoichiometry measurement and purification of
T4SS3-10+D4 subcomplexes after deletion of single T4S system components.
A SDS–PAGE analysis of purified T4SS3-10+D4 complex. Left panel: Coomassie-stained SDS–PAGE gel of the T4SS3-10+D4 complex. Right panel: Sypro Ruby-stained SDS–
PAGE gel of the T4SS3-10m and T4SS3-10+D4 complexes. Molecular weights of MW markers are shown on the left. Identification of bands is shown in the middle.
*Indicates TrwK/VirB4 degradation products. **Indicates minor contaminants OmpA and OmpC.
B Stoichiometry measurement. Fluorescence scan of an SDS–PAGE gel analysis of the T4SS3-10+D4 complex where cysteine residues were reacted with Alexa Fluor 633 C5
maleimide. Signals corresponding to the proteins coupled to Alexa Fluor 633 were detected at a wavelength of 633 nm. Fluorescent Trw protein bands are labelled,
and the derived stoichiometry for TrwB/VirD4 is indicated.
C SDS–PAGE analysis of complexes purified from cell expressing deletion mutants of the pBADM11_trwN/virB1-trwE/virB10Strep_trwD/virB11_HistrwB/virD4 constructs termed
pBADM11_DtrwX/DvirBY (see main text). The same molecular weight marker has been used on this gel as in the gel presented in panel (A). *Indicates degradation
products of TrwK/VirB4.
Source data are available online for this figure.
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TrwK/VirB4 results in the purification of a complex composed of the
core OMC components, TrwH/VirB7, TrwF/VirB9 and TrwE/VirB10, as
well as TrwB/VirD4, indicating that TrwB/VirD4 interacts directly with
the core complex, presumably through interaction with the N-
terminus of TrwE/VirB10 (Llosa et al, 2003; Segura et al, 2013). Simi-
larly, when pulling the complex using the DtrwG/virB8 construct,
apart from TrwH/VirB7, TrwF/VirB9, TrwE/VirB10 and TrwB/VirD4, only
traces of TrwK/VirB4 and its degradation products can be detected,
indicating that TrwG/VirB8 serves to stabilize TrwK/VirB4 within the
IMC. Interestingly, when the trwI/virB6 gene is not expressed with
the other T4SS components, TrwJ/VirB5 is no longer present in the
complex. This might imply an interaction between these two
components within the T4SS3-10+D4 complex and is consistent with
the known effect of VirB6 on stabilizing VirB5 in A. tumefaciens
systems (Hapfelmeier et al, 2000). TrwD/VirB11 appears to play no
role in T4SS3-10+D4 assembly as its deletion has no significant
impact on the composition of the complex purified.
These conclusions are only valid if gene deletion does not affect
expression of other genes. Although Larrea et al (2013) have
already shown using gene complementation that individual trw/virB
gene disruptions do not show any polar effect and that complemen-
tation in trans results in functional conjugative systems (except for
a disruption of trwL/virB2 that was shown to have a polar effect on
the expression of trwK/virB4), we set out to show biochemically that
deletions of single Trw/VirB proteins do not affect production and
stability of others. Thus, for each deletion constructs for which an
effect on complex formation was observed (DtrwM/virB3, DtrwK/virB4,
DtrwI/virB6 and DtrwG/virB8), we introduced sequences encoding
FLAG tags to the 50- or 30-ends of each component gene that we
observed were missing after pull-down, and monitored production
of the corresponding proteins. Since, in the DtrwM/virB3 deletion,
we observed the loss of TrwK/VirB4, TrwJ/VirB5, TrwI/VirB6 and
TrwG/VirB8, four derivatives of this DtrwM/virB3 construct were
generated in order to produce variants of these four proteins contain-
ing a FLAG tag at either their N- or C-terminus (Appendix Table S1).
Similarly, the DtrwK/virB4 deletion results in the loss of the
TrwM/VirB3, TrwJ/VirB5, TrwI/VirB6 and TrwG/VirB8 proteins: thus, we
generated four further variants of the DtrwK/virB4 construct where
these proteins were FLAG-tagged individually (Appendix Table S1).
For the DtrwI/virB6 deletion, we observed the loss of TrwJ/VirB5, and
consequently, a FLAG sequence was added to TrwJ/VirB5 and its
production checked (Appendix Table S1). Finally, deletion of
TrwG/VirB8 resulted in the destabilization of TrwM/VirB3, TrwK/VirB4,
TrwJ/VirB5 and TrwI/VirB6, and therefore, four additional constructs
of this deletion mutant were generated to include a FLAG sequence
in each of these proteins, one at a time (Appendix Table S1). To
compare production levels of each of the FLAG-tagged proteins
within deletion mutants with the amounts produced in the non-
deleted wild-type pBADM11_trwN/virB1-trwE/virB10Strep_trwD/virB11_
HistrwB/virD4 strain, FLAG sequences were also inserted in the wild-
type construct at the same position as in the deletion mutants, one
at a time (Appendix Table S1). After induction, expression was
monitored by Western blot analysis using an anti-FLAG antibody.
As can be seen in Fig EV1B, all proteins are produced. Lower
expression compared to wild type is however observed for
TrwM/VirB3 in the DtrwK/virB4 construct and for TrwI/VirB6 in the
Figure 2. Interaction between TrwB/VirD4 and TrwE/VirB10.
Left panel: Coomassie-stained SDS–PAGE gel of the T4SS3-10+D4 complex (lane 1), T4SS3-10+D4:TrwEDN42 complex (lane 2) and T4SS3-10+D4:TrwEDN64 complex (lane 3). Middle panel:
Western blot analysis using aStrep antibodies to detect the TrwE/VirB10 protein within the T4SS3-10+D4 complex and its variants. Right panel: Western blot analysis using aHis
antibodies to detect the TrwB/VirD4 protein within the T4SS3-10+D4 complex and its variants.
Source data are available online for this figure.
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The EMBO Journal VirD4 bound to a type IV secretion system Adam Redzej et al
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AC
D
B
Figure 3. NS-EM of the T4SS3-10+D4 complex.
A NS-EM micrograph of the T4SS3-10+D4 complex. A few representative particles of the T4SS3-10+D4 complex are circled in yellow. Scale bar 50 nm.
B Representative class averages of T4SS3-10+D4 complex obtained after initial alignment and classification.
C The upper row represents typical class averages of the T4SS3-10+D4 IMC complex (i.e. with the outer membrane core complex (OMC) masked out). The second row
shows projections of the T4SS3-10+D4 IMC complex in the same directions determined for the classes above. The third row displays projections of the T4SS3-10 IMC
complex (EMD-2567) in the same directions. The bottom row shows the differences between the projections above (plus or minus TrwB/VirD4) corresponding to
positions of the TrwB/VirD4 protein.
D FSC of the electron density map of the T4SS3-10+D4 complex. The 0.5 line crosses the FSC at a resolution of 28 Å.
Source data are available online for this figure.
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DtrwM/virB3 and DtrwK/virB4 constructs. Lower production of
TrwM/VirB3 and TrwI/VirB6 in these constructs might be due to an
effect of the removed protein on the stability of the TrwM/VirB3 or
TrwI/VirB6 proteins. Indeed, VirB3, 4, 6 are known to interact and
stabilize each other (Hapfelmeier et al, 2000; Mossey et al, 2010).
Nevertheless, TrwM/VirB3 and TrwI/VirB6 are produced in the
DtrwM/virB4 and DtrwK/virB3 constructs, respectively, and, although
the level of expression is lower than in the corresponding wild-type
constructs, it is similar to that observed for TrwJ/VirB5 in all
constructs including wild type, and thus cannot be considered limit-
ing. We also checked for expression of trwB/virD4 in all deletion clus-
ters and showed that each produced similar quantities of TrwB/VirD4
(Fig EV1A). We therefore conclude that the loss of particular T4SS
components during pull-down that we observe when some T4SS
components are removed is not due to the lack of expression of
these particular components. We however note that some of these
expression results contradict those reported in Larrea et al (2013):
for example, they found that production of TrwJ/VirB5 was abolished
in all insertion mutants, while, in our study, TrwJ/VirB5 is produced
in equal measure in all deletion mutants and in the wild-type
construct.
This investigation of complex formation of TrwB/VirD4 with the
T4SS3-10 complex pointed to the importance of the interaction
between TrwB/VirD4 with the core TrwH/VirB7-TrwF/VirB9-TrwE/VirB10
complex. Interaction between TrwB/VirD4 and TrwE/VirB10 has
been previously observed, and therefore, we set out to confirm
it biochemically in the context of the entire T4SS3-10+D4
complex. Within the pBADM11_trwN/virB1-trwE/virB10Strep_trwD/virB11_
HistrwB/virD4 cluster, we deleted sequences encoding either the entire
cytoplasmic tail of TrwE/VirB10 (residues 1–42; referred to as
T4SS3-10+D4:TrwED42) or both the cytoplasmic and transmembrane
segment of this protein (residues 1–64; referred to as T4SS3-10+D4:
TrwED64). After induction of expression, the T4SS3-10+D4 complex and
variants were purified and analysed by SDS–PAGE and Western blot
(Fig 2). Expression of the T4SS3-10+D4:TrwED42 resulted in lower
amounts of TrwB/VirD4 bound to the complex indicating that indeed
the cytoplasmic N-terminal region of TrwE/VirB10 is important for
interaction with TrwB/VirD4. Removal of the transmembrane segment
of TrwE/VirB10 further destabilize the interaction with the coupling
protein, confirming that the interactions between TrwE/VirB10
and TrwB/VirD4 involve the transmembrane segment of both
proteins.
Negative stain electron microscopy of the T4SS3-10+D4 complex
enables localization of the TrwB/VirD4 coupling protein within
the complex
To locate TrwB/VirD4 within the T4SS3-10+D4 complex, we solved the
structure of this complex using NS-EM and compared it to that of
the T4SS3-10 solved previously (Low et al, 2014) using the same
method (Figs 3 and 4). Since TrwD/VirB11 is not part of the
T4SS3-10+D4 complex, we produced the complex using a construct
where the trwD/virB11 gene was removed (construct DtrwD/virB11 (see
above)). Moreover, in order to prevent TrwB/VirD4 from dissociating
during sample and grid preparation, the complex was cross-linked
and re-purified using the GraFix method prior to the NS-EM analysis
(Stark, 2010). As for the T4SS3-10 complex, images of single particles
(Fig 3A) and class averages (Fig 3B) of the T4SS3-10+D4 clearly show
that the complex is made of two distinct parts, the core OMC and
the IMC, which are connected by the less defined density of the stalk
region (Fig 3B and C, upper row). A certain degree of flexibility can
also be observed in the stalk region. As TrwB/VirD4 is an inner
membrane protein, the image processing focused on the reconstruc-
tion of the IMC of the T4SS3-10+D4 complex. This was achieved to a
resolution of 28 A˚ (Fig 3D). Similarly to the T4SS3-10 IMC, two large
barrel-like densities are observed (in yellow in Fig 4). These can be
clearly divided into three layers, upper, middle and lower (Fig 4A).
They superimpose well with the two barrel-like structures identified
in the previously determined T4SS3-10 complex structure as formed
by TrwK/VirB4 trimers of dimers (Fig 4A; compare left and right
panels). Density connects the two barrels on the top, a region previ-
ously characterized as “the arches” (in green in Fig 4A–C). The
most significant differences between the structures of the T4SS3-10
and T4SS3-10+D4 IMCs are two extra regions of density present
between the two barrel-like TrwK/VirB4 structures on both sides of
the complex (Fig 3C, 2nd, 3rd and 4th rows; in blue in Fig 4A, right
panel; and Fig EV2A and B). Since the major difference in composi-
tion between the two complexes is the presence of TrwB/VirD4 in the
T4SS3-10+D4 complex, we conclude that this density must corre-
spond to TrwB/VirD4. Importantly, its shape has similarities to that
of TrwK/VirB4 in that it is also formed of three distinct layers.
TrwB/VirD4 and TrwK/VirB4 have been shown to have very similar
structures in spite of low sequence similarities (Wallden et al,
2012). Thus, it is not surprising that they should share similar
density features (Fig 4A). Interestingly, each density is not large
enough to accommodate a hexamer of TrwB/VirD4. Instead, only two
TrwB/VirD4 molecules appear to fit (Fig EV2C). However, because
negative stain is known to cause flattening and distortion, an accu-
rate evaluation of the number of molecules fitting these regions
cannot be obtained. Nevertheless, the fitting of two molecules is
consistent with the stoichiometry measurement, and therefore, it is
likely that only two TrwB/VirD4 molecules are indeed present in each
of the two regions of density we observe for this protein.
Immuno-labelling and cross-linking MS confirm the localization
of VirD4 within the T4SS3-10+D4 complex
We next sought to validate the location of VirD4 within the
T4SS3-10+D4 EM reconstruction using two complementary
techniques: immuno-labelling by NS-EM and chemical cross-linking
coupled to mass spectrometry (XL-MS).
Immuno-labelling by NS-EM provides a means by which a partic-
ular component in a large complex can be located within the elec-
tron density of that complex. In brief, an antibody targeting a
particular component of a complex is reacted to the complex; the
resulting antibody-bound complex is imaged by NS-EM. Class aver-
ages of antibody-bound complex particles are then compared to
class averages of non-bound complexes. Extra electron density
corresponding to the antibody should be observed where the anti-
body has bound and this electron density should be located within
close proximity of the component to which the antibody has bound.
To immuno-label TrwB/VirD4 within the larger T4SS3-10+D4 complex,
we generated a variant of the DtrwD/virB11 construct with a FLAG
tag-encoding sequence introduced at residue Thr236 of TrwB/VirD4.
The 236 position in the protein structure is indicated by a red sphere
in Fig 5A. This region belongs to the cytoplasmic domain of
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The EMBO Journal VirD4 bound to a type IV secretion system Adam Redzej et al
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TrwB/VirD4 and is at the opposite end of the structure relative to the
N-terminus which is known to be inserted in the inner membrane.
Therefore, the FLAG tag inserted in this region should be easily
accessible for binding by anti-FLAG antibodies and, if our localiza-
tion of TrwB/VirD4 as seen in the NS-EM structure of the T4SS3-10+D4
complex (Fig 4) is correct, the bound antibodies should be observed
on the side or at the bottom of the IMC in the NS-EM class averages
of the antibody-bound complex.
The FLAG-tagged complex termed “T4SS3-10+D4FLAG” was puri-
fied and reacted to anti-FLAG antibodies. The antibody-bound
complex was further purified, applied to a grid and stained
using negative stain as described in Materials and Methods.
After data collection, class averages were obtained: these clearly
show extra density for the FLAG antibody in close proximity of
TrwB/VirD4, either on the side or near the bottom (Fig 5B). Dif-
ference density (Fig EV3A, right panels) between class averages
of the T4SS3-10+D4 (Fig EV3A, left panels) and antibody-reacted
T4SS3-10+D4FLAG (Fig EV3A, middle panels) clearly illustrates
the presence of the extra density generated by antibody-
binding. Thus, the additional density observed by comparing
VirB7
VirB9
VirD4 VirB4
VirB8
VirB6 VirB3
VirB10
135
A
B C D
Figure 4. NS-EM structure of the T4SS3-10+D4 complex.
A Side views of the NS-EM structure of the T4SS3-10 (left) and T4SS3-10+D4 (right) complexes. The arches, the TrwK/VirB4 barrels and the TrwB/VirD4 density are shown in
green, yellow and blue, respectively. The boundaries of the three-tier structure of the TrwK/VirB4 barrel are indicated in the middle of the panels and each tier is
labelled as lower, middle and upper, respectively.
B Side view of the T4SS3-10+D4 structure (upper panel) and derived schematic representation (lower panel). Overall dimensions are indicated.
C Same as in (B) except that the side view shown is rotated 90 degrees along a vertical axis compared to the view shown in panel (B). Overall dimension is indicated.
D Bottom view of the NS-EM structure of the T4SS3-10 (upper panel) and the corresponding schematic representation (lower panel). Angle between the axes connecting
the TrwK/VirB4 hexamer pair and the TrwB/VirD4 dimer pair is shown.
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Adam Redzej et al VirD4 bound to a type IV secretion system The EMBO Journal
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the T4SS3-10 and T4SS3-10+D4 (Fig 4) can be indeed attributed to
TrwB/VirD4.
For XL-MS, the purified T4SS3-10+D4 complex was cross-linked
using amine-to-amine cross-linkers (BS3 and DSS), which connect
lysine residues that are located within a maximum distance of
33–35 A˚ (Matthew Allen Bullock et al, 2016). We used a protocol
similar to that described by Leitner et al (2014) to perform the XL-
MS experiments and applied stringent criteria during the filtering of
the database search results in order to eliminate false positives.
Using this approach, we identified a total of 41 unique cross-links
(26 inter-protein and 15 intra-protein). Of particular interest for
the study presented here are the seven cross-links involving
TrwB/VirD4 (four intra-molecular and three inter-molecular;
Appendix Table S2). Out of the four intra-molecular cross-links,
three can be mapped onto the crystal structures of R388 TrwB/VirD4
(Fig EV3B). The fourth cross-link could not be mapped onto the
A
B
C
Figure 5. Validation of the location of TrwB/VirD4 in the T4SS3-10+D4 complex.
A Location of position 236 of TrwB/VirD4 where a FLAG tag was introduced for immuno-labelling of TrwB/VirD4 within the T4SS3-10+D4 complex. Left: structure of
hexameric TrwBDN70/VirD4DN70 (PDB entry code 1GKI). Only two diametrically opposed subunits are shown. The position of residue 236 is shown with a red sphere.
Right: region of the T4SS3-10+D4 IMC where the location of the residue 236 of TrwB/VirD4 would be expected (indicated by a red sphere) and schematic representation
of an anti-FLAG antibody bound to that location.
B Class averages of antibody-bound T4SS3-10+D4FLAG. Class averages of the aligned NS-EM images of the T4SS3-10+D4FLAG with 3–4 particles per class. Red circles indicate
the position of the antibody.
C Network of spatial restraints identified by XL-MS mapped onto primary sequences of the TrwB/VirD4 subunit (blue) and the subunits interacting with it, TrwE/VirB10
(grey) and TrwK/VirB4 (yellow). Inter-molecular and intra-molecular cross-links are shown in green and orange, respectively. *Indicates cross-links mapped in the
structure shown in Fig EV3B.
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The EMBO Journal VirD4 bound to a type IV secretion system Adam Redzej et al
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structure as it involves a Lys residue in a disordered loop (PDB:
1GKI; Fig EV3B). Of the three inter-molecular cross-links, two were
between TrwB/VirD4 and TrwE/VirB10, a known interaction, and one
was with TrwK/VirB4 (Fig 5C). The observed cross-links between
TrwB/VirD4 and TrwE/VirB10 or TrwB/VirD4 and TrwK/VirB4 are consis-
tent with structural knowledge since the residues involved in the
cross-links are all likely to locate near the cytosolic side of the inner
membrane. Proximity of TrwB/VirD4 to TrwK/VirB4 was therefore
confirmed, consistent with the results of the NS-EM structure.
Conclusion
Coupling proteins in conjugative T4SSs play important roles in
recruiting the substrate to the T4S machinery (Cabezon et al, 1997;
Cascales & Christie, 2004; Lang et al, 2010, 2011). Although a lot of
information has been gathered over the years about the activity,
oligomerization state and structure as well as the importance of
VirD4 with respect to substrate recognition and processing, its loca-
tion within the T4S system was unknown (Cascales & Christie,
2004; Mihajlovic et al, 2009; Lang et al, 2010; de Paz et al, 2010;
Whitaker et al, 2016). This is the issue we set out to address in this
study. In order to achieve this, we designed functional expression
systems that resulted in TrwB/VirD4 forming a stable complex with
the previously investigated T4SS3-10 complex (Low et al, 2014). This
interaction is primarily mediated by the core OMC complex as dele-
tion of TrwM/VirB3 or TrwK/VirB4 results in the formation of a
complex between TrwB/VirD4 and the core OMC complex alone. It is
known that TrwB/VirD4 and one essential core OMC component,
TrwE/VirB10, interact, but it is nevertheless striking that this is likely
the most important interaction recruiting TrwB/VirD4 to the T4S
system as all other core OMC components are either periplasmic or
associated with the outer membrane.
The structure of the T4SS3-10+D4 sheds unprecedented light on
the location of VirD4 within the T4S machinery. Two regions of
extra electron density were present in the T4SS3-10+D4 complex,
which were absent in the T4SS3-10 complex. These densities
exhibit the three-layered structure observed for TrwK/VirB4 in the
T4SS3-10 complex (Low et al, 2014), which is to be expected as
VirD4 and VirB4 share many structural similarities (Wallden et al,
2012). The two TrwB/VirD4 patches of density are located opposite
to one another, do not connect, and the TrwB/VirD4 pair is at a
~80° angle from the pair of TrwK/VirB4 barrels (Fig 4D). Hence,
the duplication of the VirB4 ATPase observed in the T4SS3-10
complex appears to be mirrored by the VirD4 ATPase. Thus,
given that there are, in total, three different ATPases (VirB4,
VirD4 and VirB11) mediating type IV secretion, the finally assem-
bled T4S system may comprise six hexameric ATPases at any one
time, an extraordinary situation, and, to our knowledge, unique
in biology.
However, TrwB/VirD4 is not represented within our structure as a
hexamer: the electron density is only sufficient for two TrwB/VirD4
monomers (Fig EV2C). Although as an AAA+ ATPase VirD4 is
expected to form a hexamer, it has been shown previously that
TrwB/VirD4 can be purified in a monomeric form and only oligomer-
izes in the presence of DNA (Matilla et al, 2010). It has been also
hypothesized that VirD4 may bind to the T4S system as a monomer
and only oligomerizes upon substrate recruitment and binding
(Cabezon & de la Cruz, 2006). Therefore, the structure of the
T4SS3-10+D4 complex might represent the structure of the initiation
T4SS complex in its resting state, waiting for the substrate to be
delivered and activate the secretion machinery.
The close proximity of VirD4 and VirB4 might provide the most
appropriate functional platform to couple protein and DNA trans-
port. Indeed, it is known that transfer of DNA from a donor cell to a
recipient cell through the T4S system involves a covalent protein–
ssDNA complex where both, the protein and the ssDNA need to be
transferred (Zechner et al, 2012; Ilangovan et al, 2015). The
proteinaceous part of this protein–ssDNA substrate is the relaxase.
The relaxase nicks the plasmid DNA to be transferred at oriT and
covalently binds to the resulting 50-phosphate: it is this relaxase–
ssDNA complex that undergoes T4S system-mediated transport.
VirB4 has previously been described as a protein translocase while
VirD4 is thought to be involved in ssDNA transport (Atmakuri et al,
2004). It is therefore tempting to speculate that the relaxase part of
the substrate is transported through VirB4 while the ssDNA part of
the substrate is handled by VirD4: in that context, the proximity
of the two ATPases as observed in the T4SS3-10+D4 complex would
make sense.
Materials and Methods
Molecular biology
The strains and primers used in this study are listed in
Appendix Tables S3 and S4, respectively. Unless stated otherwise,
plasmids used in this study (Appendix Table S1) have been gener-
ated by following the protocol described in the IN Fusion HD
Cloning kit from Clontech.
To generate pBADM11_trwN/virB1-trwE/virB10Strep_trwD/virB11,
because the ribosome-binding site (RBS) sequence for trwD/virB11 is
contained within the sequence encoding the C-terminus of
TrwE/VirB10, a consensus Escherichia coli RBS sequence was cloned
between the stop codon for trwE/virB10 and the methionine-encoding
ATG of trwD/virB11. Using this construct, two additional clones were
produced, one with the TrwB/VirD4-encoding gene (trwB/virD4)
located after the sequence encoding TrwD/VirB11 and the other with
the TrwB/VirD4-encoding gene inserted before the sequence encoding
TrwN/VirB1. In the former, the sequence encoding a 10-Histidine tag
was added at the 50 end of the trwB/virD4 sequence so as to produce
an N-terminally His-tagged version of VirD4, while, in the latter, the
same sequence was added at the 30 end of the trwB/virD4 gene so as
to produce a C-terminally His-tagged version of the protein. Also, in
the former, expression of TrwB/VirD4 relies on its own RBS (that of
trwB/virD4) while, in the latter, expression of TrwB/VirD4 relies on the
vector’s RBS and expression of TrwN/VirB1 relies on a consensus
E. coli RBS sequence that was added. The clone with the
TrwB/VirD4-encoding sequence cloned after the TrwD/VirB11-encoding
sequence is named pBADM11_trwN/virB1-trwE/virB10Strep_trwD/virB11_
HistrwB/virD4 while the clone with the TrwB/VirD4-encoding sequence
cloned before the TrwN/VirB1-encoding sequence is named
pBADM11_trwB/virD4His_trwN/virB1-trwE/virB10Strep_trwD/virB11.
To generate the pCDF_trwABC plasmid the trwABC operon was
amplified, introducing XhoI and AscI restriction sites to the 50 and 30
ends, respectively. The PCR product was purified, digested with
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Adam Redzej et al VirD4 bound to a type IV secretion system The EMBO Journal
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XhoI and AscI, purified again and ligated into XhoI/AscI digested
pCDFDuet-1, creating the plasmid pCDF_trwABC.
To generate the pCDF_trwAC plasmid, the trwB gene was knock-
out from the pCDF_trwABC plasmid by amplification of the entire
vector except for the region encoding trwB from its 50 end to the
sequence encoding the RBS of trwC which is encoded within the
30 end of the trwB gene.
To generate the pRSForiT plasmid, the oriT region was ampli-
fied, introducing AscI restriction sites to both the 50 and 30 ends.
The PCR product was purified, digested with AscI, purified again
and ligated into AscI digested pRSFDuet-1, creating the plasmid
pRSForiT.
To generate the deletion mutants used in this study, single genes
of trwL/virB2, trwM/virB3, trwK/virB4, trwJ/virB5, trwI/virB6, trwG/virB8 or
trwD/virB11 were deleted from the pBADM11_trwN/virB1-trwE/virB10Strep_
trwD/virB11_HistrwB/virD4 plasmid. In the DtrwL/virB2 construct, the
first 35 nucleotides of the trwL/virB2 gene were maintained, as this is
the overlapping sequence of the korA gene, which is to the 50 end of
the trwL/virB2 gene. In the DtrwM/virB3 construct, the last 10 nucleo-
tides of the trwM/virB3 gene were maintained, as this sequence
contains the potential RBS sequence of the trwK/virB4 gene. In the
DtrwK/virB4 construct, the last 15 nucleotides of the trwK/virB4 gene
were maintained, as this sequence contains the potential RBS
sequence and the start codon of the trwJ/virB5 gene. In the
DtrwJ/virB5 construct, the first 4 and the last 23 nucleotides of the
trwJ/virB5 gene were maintained, as this sequence contains the stop
codon of the trwK/virB4 gene and the potential RBS sequence and the
start codon of the eex gene, respectively. In the DtrwG/virB8 construct,
the first 4 nucleotides of the trwG/virB8 gene were maintained, as
this sequence contains the stop codon of the trwH/virB7 gene.
To generate the various FLAG-tagged variants used in this study,
the pBADM11_trwN/virB1-trwE/virB10Strep_trwD/virB11_HistrwB/virD4 and
DtrwX/virBY constructs were used as templates. In order to create a
construct where TrwM/VirB3 or TrwG/VirB8 possessed a FLAG tag at
the N-terminus, the nucleotide sequence encoding the FLAG tag was
introduced at the 50 end of the trwM/virB3 and trwG/virB8 genes. In the
constructs with the FLAG tag incorporated at the C-terminus of the
TrwK/VirB4, TrwJ/VirB5 and TrwI/VirB6, the nucleotide sequence
encoding for the FLAG tag has been inserted at the 30 end of the
trwK/virB4, trwJ/virB5 and trwI/virB6 genes.
To generate the plasmid to produce TrwBDN70/VirD4DN70 for anti-
body production, the trwBDN70/virD4DN70 fragment was amplified
and inserted into pBADM11 vector.
To generate the plasmid expressing the T4SS3-10+D4FLAG complex
where a FLAG tag sequence is inserted in the loop of the TrwB/VirD4
at Thr236, the pBADM11_trwN/virB1-trwE/virB10Strep_trwD/virB11_His
trwB/virD4 vector was linearized using primers, which contained the
FLAG tag sequence.
Conjugation assay
Overnight cultures of donor cells, in this study E. coli TOP10 cells
with plasmids carrying T4S system component genes, relaxosome
genes and oriT sequence as well as recipient strain, E. coli DH5a,
were diluted and grown at 37°C with appropriate antibiotics to an
OD600 of 0.5. Expression of genes required for the DNA transfer was
induced by addition of 0.08% w/v arabinose and 1 mM IPTG. Cells
were incubated for another hour prior to performing the conjugation
assay. A total of 1.5 ml of donor culture, and the equivalent amount
of recipient culture according to OD600 measurements were washed
clean of antibiotics using LB media and filtered on a hydrophilic
MF-Millipore Membrane (Merck Millipore), made of mixed cellulose
esters with a pore size of 0.45 lm. The filters were then incubated
for 1.5 h on a LB agar plate with cells facing upwards. The cells
were subsequently washed off the filter paper using 1 ml LB, and
100 ll of different dilutions was plated on LB agar plates containing
nalidixic acid (Nal, selecting for E. coli DH5a recipient strains) and
a second antibiotic, whose resistance is encoded on the transported
plasmid (kanamycin (Kan) in this study). Appropriate dilutions of
the conjugation culture were also plated on antibiotics selecting for
donors or total E. coli DH5a recipients. The transfer efficiency was
calculated by dividing transconjugant colony count by recipient
colony count (Cellini et al, 1997).
Purification of the T4SS3-10+D4 complex, single-component
deletion mutants and FLAG-tagged T4SS3-10+D4FLAG variant
The T4SS3-10+D4 complex or the single-component deletion mutants
of the T4SS3-10+D4 complex were purified according to the following
procedure. Overnight culture of a freshly transformed TOP10 cells
(Thermo Fischer) were diluted into six litre of LB medium supple-
mented with 100 lg/ml of carbenicillin. Cells were grown at 37°C
with shaking. When the OD600 of the cell culture reached 0.5, cells
were induced by addition of 0.08% arabinose and the expression
was carried on at 16°C with shaking overnight. Cells were pelleted
by spinning down for 30 min at 5,000 × g and resuspended in lysis
buffer: 50 mM HEPES pH = 7.6, 200 mM sodium acetate, 1 mM
EDTA pH = 8.0 with a protease inhibitor cocktail tablet (Roche),
100 lg/ml lysozyme and 1 lg/ml DNaseI. After stirring of the
mixture on ice for 15 min, the cells were lysed by passing them
twice through an Emulsiflex C-5. Cell debris was separated from the
supernatant by spinning for 30 min at 26,712 × g. Membranes were
pelleted by ultracentrifugation of the supernatant for 45 min at
95,834 × g and manually homogenized using solubilization buffer:
50 mM HEPES pH = 7.6, 200 mM sodium acetate, 1 mM EDTA
pH = 8.0, 0.5% w/v n-dodecyl-b-D-maltopyranoside (DDM) (Ana-
trace), 0.5% digitonin (Sigma-Aldrich), 0.05 mM n-tetradecyl-N,N-
dimethylamine-N-oxide (TDAO) (Anatrace), 2 mM tris(2-carboxy-
ethyl)phosphine (TCEP) (Sigma-Aldrich) and placed on the rotary
shaker at 4°C for 1 h. Insoluble materials were separated by another
ultracentrifugation for 20 min at 95,834 × g. The supernatant was
loaded to a 5 ml Strep column (GE Healthcare) equilibrated with
buffer A: 50 mM HEPES pH = 7.6, 200 mM sodium acetate, 0.1%
digitonin, 0.05 mM TDAO. After extensive washing with buffer A,
samples were eluted with buffer A + 2 mM desthiobiotin and
50 mM imidazole pH = 7.6 directly into 2 × 1 ml HisTrap columns
(GE Healthcare) equilibrated with the same buffer. After additional
washing step with buffer A containing 100 mM imidazole pH = 7.6,
the protein complex was eluted with buffer A containing 350 mM
imidazole pH = 7.6.
Sample of the T4SS3-10+D4 complex used for the NS-EM experi-
ments was in addition to the protocol mentioned above stabilized
by mild cross-link with 0.1% of glutaraldehyde (Sigma-Aldrich)
while being applied to the 10–30% sucrose density gradient at
50,512 × g for 15 h at 4°C using a SW40Ti rotor (Stark, 2010).
Samples from the gradient were fractionated and analysed by
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The EMBO Journal VirD4 bound to a type IV secretion system Adam Redzej et al
3090
SDS–PAGE. Fractions with bands at high molecular weight on the
gels were analysed by NS-EM. Grids of the samples which looked
the most concentrated and least aggregated on the NS-EM were
used for collection of the images which were used for EM data
processing.
Electron microscopy and data processing
Negative stain electron microscopy (NS-EM) was used to analyse
the complex. 5 ll of T4SS3-10+D4 complex at concentration of
around 0.03 mg/ml was applied on glow discharged carbon coated
copper grids (type B 400, Agar Scientific) and incubated at room
temperature (RT) for 3 min. The excess of the sample was blotted
away and the grid was stained with 5 ll of 2% uranyl acetate for
3 min. The excess stain was then blotted away and the grid was
dried at room temperature. Grids were imaged on a Tecnai F20 FEG
microscope operating at 200 kV at a magnification of 41,500 using a
defocus range of 0.5 to 2 lm. Images were recorded using the
direct electron detector DE20 (1.54 A˚ per pixel) using a total dose of
30 electrons/A˚2. Frames from 2 to 29 were aligned using EMAN2
(Tang et al, 2007) and IMOD (Kremer et al, 1996) software. The
CTF was estimated using CTFFIND3 (Mindell & Grigorieff, 2003)
and correction was done by phase flipping using BSOFT (Heymann,
2001) on the aligned frames. A total of 14,918 particles were manu-
ally selected using XMIPP v3.0 (Scheres et al, 2008) and extracted
from aligned, averaged and CTF corrected frames with a box size of
400 pixels. All subsequent operations were done using IMAGIC-5
(van Heel et al, 1996).
Images were normalized, band-pass filtered and subjected to
multireference alignment (MRA) using re-projections (side views
only) of the 3D reconstruction EMD-2567 of the T4SS3-10 complex,
followed by multivariate statistical analysis (MSA) (van Heel et al,
2000). The best classes representing characteristic views were used
as new references for the next iteration of the alignment procedure.
Aligned particle images were shifted up so the centre of the IMC
was located in the centre of the frame, then a circular mask was
applied to remove parts of the image corresponding to the outer
membrane core complex and the surrounding noise. Then several
rounds of MRA and MSA were run using re-projections of the IMC
part of the T4SS3-10 reconstruction (i.e. EMD-2567 where the outer
membrane core complex was masked out). The best classes were
selected according to the minimal deviation between images that
compose the classes. Euler angles were assigned to these classes by
angular reconstitution (Van Heel, 1987) using an anchor set
calculated using the IMC part of the T4SS3-10 reconstruction. No
symmetry was applied during the first steps of the reconstruction.
The first asymmetrical reconstruction clearly demonstrated features
corresponding to C2 symmetry with a self cross-correlation of the
non-symmetrized map of about 75%. During subsequent steps of
refinement, the C2 symmetry was applied. To improve the align-
ment and angular assignment further rounds of MRA-MSA-angular
reconstitution were run iteratively using the best classes for the
reconstructions. Eventually, 2000 classes were used with around 7-8
particles per class during the refinement procedure. In the final
structure, ~700 classes with ~4 images per class were used. The
resolution of the 3D reconstruction was estimated by Fourier
Shell Correlation (FSC) (van Heel & Schatz, 2005) as 28 A˚ using
0.5 criteria. Visualization of the final 3D map of the IMC was made
in Chimera (Pettersen et al, 2004). Segmentation of the T4SS3-10+D4
complex was performed using Chimera.
Determination of the complex stoichiometry by cysteine labelling
with Alexa Fluor dye
50 ll (~0.3 mg/ml) of sample of the T4SS3-10+D4 complex after
elution from the HisTrap column was buffer exchanged using a PD
10 (GE Healthcare) desalting column to a buffer containing 50 mM
HEPES pH 7.2, 200 mM sodium acetate and 1 mM EDTA. The
complex was then denaturated by boiling in 1% SDS. Disulphide
bonds were reduced by incubation with TCEP at 2 mM concentra-
tion for 20 min at room temperature. Alexa Fluor 633 C5 malei-
mide (Thermo Fischer Scientific) was added to 200 lM final
concentration and samples were incubated at room temperature for
an additional 1 h protected from light. Proteins were separated
using SDS–PAGE, and the gel was visualized using Image Reader
FLA-3000. Intensity of the bands was analysed using the Image
Gauche software.
Immuno-labelling of the T4SS3-10+D4FLAG complex and
visualization by NS-EM
T4SS3-10+D4FLAG complex was purified using the same protocol as
described for the T4SS3-10+D4 complex. Freshly prepared
T4SS3-10+D4FLAG sample was incubated with anti-FLAG antibody
produced in goat (Abcam) in a 1:5 molar ratio for 1 h on ice. The
excess of the antibody was removed by loading the mixture to a
1 ml StrepHP column (GE Healthcare) equilibrated with buffer A.
After washing the column with buffer A the complex with the anti-
body was eluted from the column using buffer A + 2 mM desthio-
biotin. The presence of the antibody within the eluted complex was
confirmed by Commasie stained SDS–PAGE gel and WB, using anti-
goat antibodies conjugated with alkaline phosphatase (Abcam). The
bands were visualized by incubating the membrane with SIGMA-
FASTTM BCIP/NBT (Sigma-Aldrich).
NS-EM grids of the T4SS3-10+D4FLAG with the anti-FLAG antibody
were prepared as described for the T4SS3-10+D4 complex. Grids were
imaged on a Tecnai F20 FEG microscope operating at 200 kV at a
magnification of 35 750 using defocus range 1.5 to 2 lm. Images
were recorded using the direct detector DE20. Movies were collected
(1.79 A˚ per pixel) using a total dose of 30 electrons/A˚2. Frames from
2 to 20 were aligned using EMAN2 (Tang et al, 2007) and IMOD
(Kremer et al, 1996) software. The CTF was estimated using
CTFFIND3 (Mindell & Grigorieff, 2003) and correction was done by
phase flipping using BSOFT (Heymann, 2001) on the aligned
frames. A total of 80 particles, representing complexes with and
without a clear extra density corresponding to antibody, were
manually picked from aligned, averaged and CTF corrected frames
with a box size of 320 pixels. All subsequent operations were done
using IMAGIC-5 (van Heel et al, 1996).
Images were normalized and aligned to one of the images. This
was followed by MSA (van Heel et al, 2000). Next the images were
classified based on the eigenvectors which showed the differences
corresponding to the presence and absence of the antibody within
the particles. To identify the binding site of the antibody within the
complex, six classes with 3–4 particles per class were selected. Class
averages of the particles with the antibody bound were compared
ª 2017 The Authors The EMBO Journal Vol 36 | No 20 | 2017
Adam Redzej et al VirD4 bound to a type IV secretion system The EMBO Journal
3091
with particles within the same orientation to localize the extra
density corresponding to the antibody. The difference between the
two has been created as well.
Purification of TrwBDN70/VirD4DN70 for raising antibodies
Overnight culture of a BL21* cells (Thermo Fisher) freshly trans-
formed with pBADM11His_TEVtrwB/virD4DN70 (Appendix Table S1)
was diluted into six litre of LB medium supplemented with appropri-
ate antibiotic. Cells have been grown at 37°C with shaking. When
the OD600 of the cell culture reached 0.5, cells were induced by the
addition of 0.08% arabinose and the expression was carried out at
16°C with shaking overnight. Cells were pelleted by spinning down
for 30 min at 5,000 × g and resuspended in lysis buffer: 50 mM
HEPES 7.6, 250 mM NaCl, 1 mM EDTA with protease inhibitor
cocktail tablet (Roche), 100 lg/ml lysozyme and 1 lg/ml DNaseI.
After stirring of the mixture on ice for 15 min, the cells were lysed
by passing them twice through Emulsiflex C-5. Cell debris was sepa-
rated from the supernatant by ultracentrifugation for 45 min at
235,418 × g. Supernatant was filtered through 0.45-lm filter and
loaded to the 5 ml HisTrap column equilibrated with loading buffer
(50 mM HEPES 7.6, 250 mM NaCl, 100 mM imidazole). After wash-
ing the column proteins were eluted with an increasing concentra-
tion of imidazole up to 500 mM imidazole. Fractions containing the
protein were pooled and dialysed overnight to the loading buffer.
During the dialysis, the protein sample was incubated with 6xHis-
TEV protease in mass ratio 1:50 of the protease to the protein.
Cleaved protein was separated from the protease and the non-
cleaved protein by passing the sample through a 5 ml HisTrap
column equilibrated with loading buffer. Flow through from the
column was collected and concentrated by dialysis to PBS buffer
adjusted with 50% glycerol. Proteins were aliquoted and flash
frozen and stored at 80°C.
Antibody purification
Antibodies were purified based on manufacturer’s protocol. Briefly,
1 ml HiTrap NHS-activated HP column (GE Healthcare) was acti-
vated by applying a drop of ice cold 1 mM HCl followed by slowly
washing it with additional 6 ml of 1 mM HCl. 1 ml of protein
sample (0.7 mg of TrwBDN70/VirD4DN70), which has been buffer
exchanged into coupling buffer (0.2 M NaHC03, 0.5 M NaCl,
pH = 8.3), was immediately loaded to the column and incubated at
room temperature for 30 min. Excess of uncoupled protein was
removed by washing the column with 1 ml of coupling buffer.
Column was then deactivated by sequential washes with 6 ml of
buffer A (0.5 M ethanoloamine pH = 8.3), 6 ml of buffer B (0.1 M
sodium acetate pH = 4.0, 0.5 M NaCl) and another 6 ml wash with
buffer A. Column with protein was incubated at this step at room
temperature for 30 min. Afterwards, the column was washed with
6 ml of buffer B, 6 ml of buffer A, 6 ml of buffer B and finally with
5 ml of PBS buffer. 20 ml of rabbit blood serum containing antibod-
ies was loaded to the column overnight at 0.1–0.2 ml/min flow rate.
The column was afterwards washed with 40 ml of PBS followed by
additional 20 ml wash with PBS supplemented with 0.5 M NaCl and
another 20 ml wash with PBS buffer. Antibodies were eluted from
the column using elution buffer (100 mM glycine pH = 2.3). In
order to immediately neutralize the very low pH of the buffer that
the samples are eluted in, 2 M Tris pH = 8.0 was added to the
collection tubes before starting the fractionation. Fractions were
analysed on the SDS–PAGE gel and the samples containing anti-
bodies were pooled and dialysed against PBS buffer supplemented
with 50% glycerol.
Detection of the expression levels of TrwB/VirD4 within the
different deletion mutants expressing R388 T4S system
Overnight culture of TOP10 strain freshly transformed with a plas-
mid carrying genes expressing either T4SS3-10+D4 or T4SS3-10+D4
lacking one of the T4S complex encoding genes were diluted into a
LB medium supplemented with 100 lg/ll of carbenicillin and
grown and 37°C at 185 rpm. When the OD600 of the cultures
reached 0.5, the expression of the genes was induced by addition of
Arabinose at 0.08% w/v concentration. The expression was carried
out at 18°C overnight. The equivalent of 1 ml of OD600 1 was
collected from each sample and the cells were spun down for 5 min
at 16,100 × g. Next, the cells were resuspended in NuPAGE LDS
Sample Buffer (Thermo Fischer) and lysed by passing them 15 times
through a syringe equipped with a 0.5-lm needle. Sample was
boiled at 95°C for 5 min and cooled down on ice before loading to
the SDS–PAGE gel. In order to ensure that the signal quantified from
the Western blot is in the linear range, samples of purified
TrwBDN70/VirD4DN70 at known concentration were loaded on the
same gel as the samples of the overexpressed variants of the R388
T4S system. The antibodies used to detect the amount of expressed
TrwB/VirD4 from different constructs were TrwBDN70 antibody
raised in rabbit and isolated in house in combination with rabbit
anti-goat IgG (Thermo Fisher). The bands were visualized by incu-
bating the membrane with SIGMAFASTTM DAB with metal enhancer
(Sigma-Aldrich).
Detection of the expression levels of FLAG-tagged T4SS
components within the different deletion mutants expressing
R388 T4S system
The samples have been prepared in the same way as described
above for the detection of the TrwB/VirD4 expression. The only dif-
ference being that the expressed proteins were visualized by incu-
bating the membrane with anti-FLAG antibody produced in rabbit
(Abcam) followed by incubation with anti-rabbit antibody conju-
gated with horseradish peroxidase (Abcam). The bands were visual-
ized by incubating the membrane with SIGMAFASTTM DAB with
metal enhancer (Sigma-Aldrich).
Cross-linking-mass spectrometry
Chemical cross-linking of the purified T4SS3-10+D4 complex
50–150 lg of purified T4SS3-10+D4 (at 0.1–0.5 mg/ml) was cross-
linked either with 2.5–5 mM BS3 d0/d12 mixture (Creative Mole-
cules; dissolved freshly in 20 mM HEPES pH 7.6 at 50 mM) or
1–1.5 mM DSS d0/d12 mixture (Creative Molecules; dissolved
freshly in DMF at 25 mM) at RT for 40 min with mild shaking. The
reaction was quenched by addition of ammonium bicarbonate
solution to a final concentration of 50 mM and the solvent was
evaporated using a vacuum centrifuge. The cross-linking was
confirmed by SDS–PAGE and the cross-linked sample was examined
The EMBO Journal Vol 36 | No 20 | 2017 ª 2017 The Authors
The EMBO Journal VirD4 bound to a type IV secretion system Adam Redzej et al
3092
by negative staining electron microscopy to ensure that the sample
had not aggregated during the cross-linking reaction. The final set of
cross-links was collected from six independent purifications.
Sample preparation for LC-MS/MS
The cross-linked sample was prepared for LC-MS/MS using the
following procedure (modified from Leitner et al, 2014): The sample
was denatured by dissolving it in 8 M urea and 0.1% Rapigest
(Waters) at 1 mg/ml. Disulphide bonds were reduced by 10 mM
DTT at 37°C for 30 min and free cysteines were alkylated by 20 mM
iodoacetamide at RT in the dark for another 30 min. The sample
was then incubated with Lys-C (Wako Chemicals GmbH) at 1:50
(w/w) enzyme-to-substrate ratio for 2 h at 37°C with mild shaking.
The sample was then diluted with 50 mM ammonium bicarbonate
solution to a final concentration of 1 M urea and trypsin (Promega)
was added at an enzyme-to-substrate ratio of 1:50 (w/w). The
mixture was incubated overnight at 37°C with mild shaking. Follow-
ing overnight digestion, the sample was acidified by adding formic
acid to a final concentration of 2% (v/v). The sample was purified
by solid-phase extraction (SPE) using 50 mg Sep-Pak tC18 cartridges
(Waters). Briefly, the cartridge was activated with 500 ll of acetoni-
trile (ACN) and equilibrated with 1 ml of SPE wash solution (water/
ACN/formic acid: 95/5/0.1 (v/v/v)). After loading the sample, the
cartridge was washed twice with 500 ll of SPE wash solution and
the sample was eluted with 500 ll of SPE elution solution (water/
ACN/formic acid: 50/50/0.1 (v/v/v)). The eluted sample was
concentrated by removing the solvent using a vacuum centrifuge.
Following SPE, the sample was dissolved in 20 ll of SEC buffer
(water/ACN/TFA: 70/30/0.1 (v/v/v)) and the cross-linked peptides
were enriched using size-exclusion chromatography (SEC) by sepa-
ration of peptides on a Superdex Peptide PC 3.2/300 column (pre-
equilibrated with SEC buffer) using an AKTA Micro system. 100 ll
collected fractions were concentrated by removing the solvent using
a vacuum centrifuge. The SEC fractions were dissolved in MS buffer
(water/ACN/TFA: 97/3/0.1 (v/v/v)) and analysed by LC-MS/MS.
LC-MS/MS analysis
The collected fractions were analysed by LC-MS/MS using a
nanoAcquity UPLC system (Waters) connected to LTQ-Orbitrap
Velos instrument (ThermoFisher Scientific). Peptides were separated
on an NTCC-360/100-5-153 column (Nikkyo Technos Co.) at a flow
rate of 400 nl/min using the following gradient: 0–60 min, 3–40%B;
60–61 min, 40–85%B; 61–71 min, 85%B, where solvent A = 0.1%
(v/v) formic acid in water and B = 0.1% (v/v) formic acid in ACN.
The MS spectra were acquired in the Orbitrap (m/z range 400–
2,000; 60,000 resolution), and up to 10 most abundant ions per scan
were selected for fragmentation by collision-induced dissociation
(normalized collision energy = 35, activation Q = 0.250) and anal-
ysed in the linear ion trap. Precursors of unknown charge state or
charge states +1 and +2 were not selected for fragmentation, and
selected precursors were added on a dynamic exclusion list for 10 s
after one sequencing event. Samples were analysed in duplicates.
XL-MS data analysis
Raw files were converted to the mzXML format using msconvert
(Chambers et al, 2012) and then searched using xQuest (Leitner
et al, 2014) against a database containing the sequences of VirB1 to
VirB11 and VirD4. False discovery rates (FDR) were estimated using
xProphet, and the results were filtered using the following parame-
ters: FDR = 0.05, min delta score = 0.95, MS1 tolerance window =
10 ppm, Id-score > 25, minimum number of cleavages per
peptide = 3.
Accession codes
The EM map of the T4SS3-10+D4 complex has been deposited in the
EMD data bank (http://www.ebi.ac.uk/pdbe/emdb) under EMDB
entry code EMD-3585.
Expanded View for this article is available online.
Acknowledgements
This work was supported by Wellcome Trust 098302 grant to GW. We would
like to thank Prof Jasminka Godovac-Zimmermann and Dr Nigel Rendell for
providing us access to the Orbitrap mass spectrometer used for the cross-
linking experiments.
Author contributions
AR, SC and GW designed the experiments; AR, MT and SC cloned all the
constructs used in this study and AR and SC checked their functionality for
DNA transfer; AR and MU purified the T4SS3-10+D4 complexes and variants and
determined stoichiometry; MU collected the NS-EM data set of the T4SS3-10+D4
complex and together with EVO solved the structure of the T4SS3-10+D4
complex; EVO supervised the EM analysis; AR performed the immuno-labelling
experiments; AR and MT purified TrwB used for raising of the antibodies and
purified the antibodies from the obtained serum; AR and CF-R purified the
deletion constructs; MT performed XL-MS experiments and analysed the data
with the help from AC and KT; AR, SC, RDH and GW wrote the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
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