Downloaded from http://rstb.royalsocietypublishing.org/ on July 31, 2017
rstb.royalsocietypublishing.org
Insertion of proteins and
lipopolysaccharide into the bacterial
outer membrane
Istvan Botos1, Nicholas Noinaj2 and Susan K. Buchanan1
Review
Cite this article: Botos I, Noinaj N, Buchanan
SK. 2017 Insertion of proteins and
lipopolysaccharide into the bacterial
outer membrane. Phil. Trans. R. Soc. B 372:
20160224.
http://dx.doi.org/10.1098/rstb.2016.0224
Accepted: 12 October 2016
One contribution of 17 to a discussion meeting
issue ‘Membrane pores: from structure and
assembly, to medicine and technology’.
Subject Areas:
structural biology, microbiology
Keywords:
BAM complex, BamABCDE, outer-membrane
biogenesis, LptDE, lipopolysaccharide
Author for correspondence:
Susan K. Buchanan
e-mail: [email protected]
Electronic supplementary material is available
online at https://dx.doi.org/10.6084/m9.
figshare.c.3775496.
1
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, MD 20892, USA
2
Markey Center for Structural Biology, Department of Biological Sciences, and the Purdue Institute for
Inflammation, Immunology and Infectious Diseases, Purdue University, West Lafayette, Indiana 47907, USA
SKB, 0000-0001-9657-7119
The bacterial outer membrane contains phospholipids in the inner leaflet and
lipopolysaccharide (LPS) in the outer leaflet. Both proteins and LPS must
be frequently inserted into the outer membrane to preserve its integrity. The
protein complex that inserts LPS into the outer membrane is called LptDE,
and consists of an integral membrane protein, LptD, with a separate globular
lipoprotein, LptE, inserted in the barrel lumen. The protein complex that inserts
newly synthesized outer-membrane proteins (OMPs) into the outer membrane
is called the BAM complex, and consists of an integral membrane protein,
BamA, plus four lipoproteins, BamB, C, D and E. Recent structural and functional analyses illustrate how these two complexes insert their substrates into
the outer membrane by distorting the membrane component (BamA or
LptD) to directly access the lipid bilayer.
This article is part of the themed issue ‘Membrane pores: from structure
and assembly, to medicine and technology’.
1. Introduction
A cell envelope consisting of an inner membrane, a periplasmic space, and an
outer membrane protects Gram-negative bacteria from environmental stresses
and toxins. While phospholipids make up both leaflets of the inner membrane,
the outer membrane is an asymmetric structure where the inner leaflet contains
phospholipids, while the outer leaflet consists of lipopolysaccharides (LPS) coordinated by divalent metal ions such as calcium and magnesium. The periplasmic
space separating the inner and outer membranes accommodates a layer of
peptidoglycan, and has been estimated to have an average thickness of 150 Å [1].
To maintain the integrity of the outer membrane, both LPS and certain
outer-membrane proteins (OMPs) are required for viability, and they must be
transported across the cell envelope and positioned correctly in the outer membrane. As described in the following sections, both proteins and lipids are
thought to be inserted laterally into the outer membrane through openings in
the protein and LPS biogenesis machineries. Lateral migration of lipids and proteins is a common theme that has been observed before [2]. The lateral
movement of proteins into a lipid bilayer has been demonstrated for the Sec
translocon, where individual hydrophobic, membrane-spanning a-helices are
co-translationally translocated into the inner membrane, where the final
assembly of a-helical inner membrane proteins takes place [3,4]. Similarly,
two b-barrel OMPs can transfer hydrophobic substrates from the lumen of
the b-barrel to the outer membrane. FadL binds long-chain fatty acids first at
a low-affinity (extracellular) site, and then the substrate is moved by diffusion
to a high-affinity site within the barrel lumen. A conformational change
occurs to release the substrate from the high-affinity site and provides free
access to a ‘hole’ in the barrel wall, allowing the substrate to diffuse laterally
into the bilayer through the barrel opening [5–7]. A second example is found
& 2017 The Author(s) Published by the Royal Society. All rights reserved.
Downloaded from http://rstb.royalsocietypublishing.org/ on July 31, 2017
LPS
BamA
OMP
OM
LptE
P5
BamC BamD
BamE
P3
BamB
phospholipid
LptA
P2
P1
BAM complex
SurA
LptC
periplasm
LptF
Sec
LptG
MsbA
IM
LptB LptB
cytoplasm
ATP
nascent OMP
ATP
ADP + Pi
ADP + Pi
Figure 1. Transport of LPS and nascent OMPs to the outer membrane. Schematic pathways are shown for the biogenesis of b-barrel outer-membrane proteins and
lipopolysaccharide in Gram-negative bacteria.
in PagP, an outer-membrane acyltransferase that protects bacteria against the host immune response by transferring a
palmitate chain from a phospholipid to Lipid A. When substrate binds, His, Asp and Ser residues located on the
extracellular surface of the barrel facilitate transfer of the palmitoyl group from the donor phospholipid to the Lipid A
acceptor molecule [8,9].
In the outer membrane, the only essential OMPs in almost
all Gram-negative bacteria are the biogenesis machineries
that function to insert LPS and OMPs. This review describes
recent structural and functional studies that illustrate the
complex processes of protein and LPS insertion into the
outer membrane.
they form a continuous protein network spanning the entire
cell envelope (figure 1). LptB2FG is an ABC transporter that
hydrolyses ATP to provide energy for LPS extraction from
the inner membrane [13], where it is transferred to LptC
[14]. LptC is thought to interact with a polymer chain of
LptA molecules that span the periplasmic space, with the
terminal LptA linking to the periplasmic domain of LptD
in the outer membrane [15]. LptA, C and D share a
common fold with a hydrophobic groove that is predicted
to shield Lipid A from the aqueous environment of the
periplasm during transit. The Lpt components thus form a
trans-envelope scaffold that drives transport of LPS by hydrolysing ATP at the inner membrane [16,17]. Once it reaches
LptD, the LPS molecule is inserted into the outer membrane
by the LptDE complex [18].
2. The lipopolysaccharide-transporting complex
LptDE
The biogenesis pathway for LPS has been elegantly defined
in recent years [10]. LPS consists of three components:
Lipid A contains the hydrophobic hydrocarbon chains that
anchor the molecule in the outer membrane, while a core
oligosaccharide and a variable O-antigen carbohydrate
chain, containing up to 200 sugars, are hydrophilic and eventually reside on the cell surface [11]. Both the Lipid A and
oligosaccharide molecules are independently synthesized in
the cytoplasm and translocated to the outer leaflet of the
inner membrane, where the bulky O-polysaccharide is
assembled on the Lipid A core, yielding mature LPS [11].
Mature LPS is then extracted from the inner membrane,
transported across the periplasmic space, and inserted into
the outer membrane by the LPS transport system LptABCDEFG [12]. All seven Lpt components are essential because
3. Description of the LptDE structures
Four LptDE structures were recently determined: the structure of full-length Klebsiella pneumoniae LptDE, and three
N-terminally truncated LptDE core complexes from
K. pneumoniae, Pseudomonas aeruginosa and Yersinia pestis
[19]. In addition, a full-length LptDE structure was determined from Shigella flexneri [20] and a core complex from
Salmonella typhimurium [21], as well as a core complex from
Escherichia coli (PDB code 4RHB). With 26 b-strands, LptD
is the largest known single-protein b-barrel. Also, it is the
only protein to use a separate lipoprotein, LptE, as its
‘plug’ domain. Most of the globular lipoprotein LptE is
nested in the barrel lumen and around a quarter of its surface
is exposed to the periplasm. This architecture is preserved in
Phil. Trans. R. Soc. B 372: 20160224
chaperones
P4
rstb.royalsocietypublishing.org
LptD
2
Downloaded from http://rstb.royalsocietypublishing.org/ on July 31, 2017
3
rstb.royalsocietypublishing.org
LptD
barrel
b26
LptD
N-terminal
domain
LptE
Figure 2. Structure of the Klebsiella pneumoniae full LptDE complex. The LptD b-barrel is shown in orange, with the N-terminal domain in green. The LptE plug is
shown in blue. The first and last b-strands of the barrel are highlighted in red.
all currently determined LptDE crystal structures (figure 2
and table 1).
LptD and LptE interact very strongly with one another.
These two proteins co-purify as a complex from detergentsolubilized outer membranes, they migrate as heterodimers
on size exclusion columns, and are resistant to dissociation
in solution, except when heated. The co-purified complex
also migrates as a single band on a denaturing gel [18,35].
The dimensions of the elliptical transmembrane LptD
b-barrel are around 50 50 70 Å, with three to five hydrogen bonds between the first (b1) and last (b26) strands
closing the b-barrel. Strand b1 of the barrel is preceded by
a periplasmic b-jellyroll domain consisting of two 11-strand
b-sheets arranged in a V shape that form a hydrophobic
groove, homologous to LptA. Terminal strand b26 contains
a short C-terminal extension that resides in the lumen of
the b-barrel. The b-barrel is also slightly tapered toward the
extracellular side, with its periplasmic diameter around
10 Å larger than its extracellular diameter. LptE exhibits an
a/b fold with two a-helices and five b-strands. Despite low
sequence identity, individual LptE crystal structures from
Shewanella oneidensis (PDB code 2R76), Caulobacter crescentus
(PDB code 4KWY), Neisseria meningitidis (PDB code 3BF2),
Escherichia coli (PDB code 4NHR) [25] and the solution structures from Nitrosomonas europeae (PDB code 2JXP) and
Pseudomonas aeruginosa (PDB code 2N8X) [28] are almost
identical to their LptDE-complexed counterparts (table 1).
LptE has several distinct functions: assembly of the LptD
b-barrel, stabilization of the LptDE complex by forming a
plug and LPS translocation [18,25,36 –38]. The N-terminus
of LptE is anchored to the inner leaflet of the outer membrane
by an N-acyl-S-diacylglycerylcysteine moiety that is not
visible in any of the crystal structures. While the b-barrel
lumen is mainly hydrophilic, the LptE surface has both
hydrophobic and hydrophilic patches, making it highly
unlikely to bind the hydrophobic portion of LPS. The size
of the LptDE lumen is large enough to accommodate an
Ra LPS molecule (Ra LPS contains Lipid A, an inner core
of oligosaccharide consisting of at least one molecule of
3-deoxy-D-manno-oct-2 ulosonic acid (KDO) linked to Lipid
A, and an outer core of oligosaccharide) [20].
The available LptDE structures exhibit an identical fold,
with the Pseudomonas LptDE structure being slightly different
due to two insertion loops. These insertion loops are located
in the extracellular loop region of the barrel, conferring a
larger molecular surface to the Pseudomonas LptDE core complex [19]. The significance of the larger molecular surface of
LptDE for this species is not clear, but may be related to
the different O-antigen structure of Pseudomonas LPS.
4. Insertion mechanism
LPS translocation differs from protein translocation by BamA
[32] or long-chain fatty acid transport by FadL [39]. The bbarrel opens only partially between strands b1 and b26,
forming a lateral gate that permits diffusion of the Lipid A
domain of LPS into the outer leaflet of the outer membrane.
This can be attributed to the weakened hydrogen bonding
interactions between strands b1 and b26 caused by two proline residues, P231 and P246 [20]. Mutation of these proline
residues yields reduced viability in in vivo assays and closer
association of the b1 and b26 strands in MD simulations
[19]. Double cysteine mutations that link b1 to b26, and
Phil. Trans. R. Soc. B 372: 20160224
b1
Downloaded from http://rstb.royalsocietypublishing.org/ on July 31, 2017
Table 1. Table of Lpt and BAM structures discussed in the manuscript.
organism
resolution (Å)
PDB code
reference
LptE and LptDE complexes
Klebsiella pneumoniae
Klebsiella pneumoniae
2.94
4.37
5IV8
5IV9
[19]
[19]
LptDE core
LptDE core
Pseudomonas aeruginosa
Yersinia pestis
2.99
2.75
5IVA
5IXM
[19]
[19]
LptDE core
LptDE full complex
Salmonella enterica
Shigella flexneri
2.8
2.39
4N4R
4Q35
[21]
[20]
LptDE core
Escherichia coli
3.36
4RHB
[22]
LptE
LptE
Caulobacter crescentus
Neisseria meningitidis
2.4
2.6
4KWY
3BF2
[23]
[24]
LptE
LptE
Esherichia coli
Shewanella oneidensis
2.34
2.6
4NHR
2R76
[25]
[26]
LptE
Nitrosomonas europea
NMR
2JXP
[27]
LptE
BamA and BAM complexes
Pseudomonas aeruginosa
NMR
2N8X
[28]
Bam ACDE
Bam ABCDE
Escherichia coli
Escherichia coli
3.39
2.9
5EKQ
5D0O
[29]
[30]
Bam ACDE
Bam ABCDE
Escherichia coli
Escherichia coli
3.5
3.56
5D0Q
5AYW
[30]
[31]
Bam A (þ2 POTRA)
Haemophilus ducreyi
2.91
4K3C
[32]
Bam A (þ5 POTRA)
Bam A (þ1 POTRA)
Neisseria gonorrhoeae
Escherichia coli
3.2
3.0
4K3B
4C4 V
[32]
[33]
Bam A (barrel only)
Escherichia coli
2.6
4N75
[34]
block the opening of the lateral gate, are lethal to the bacteria
[21]. The LptDE lateral gate opening is also limited by the
presence of the LptD N-terminal domain and the conserved
disulfide bond between the b24-b25 turn and the N-terminal
domain. This N-terminal domain can rotate up to 218 around
the hinge region that connects it to the b-barrel, as observed
by comparing the two full-length LptDE structures [19,20]
and it can physically block LPS from entering the inner leaflet
of the outer membrane.
The tapering lumen of the LptD barrel is lined by conserved, increasingly electronegative residues that create
unfavourable interactions with the negatively charged sugar
moieties of the LPS molecule. Once the b-barrel outer loops
open and the O-antigen portion of LPS is translocated to the
outer leaflet of the outer membrane, the charge repulsion is
resolved. Mutagenesis of the lumen residues showed that a
strongly electronegative lumen is not required for transport,
but an electropositive lumen does inhibit LPS translocation
[19]. Recent studies using UV photoaffinity cross-linking
delineate steps in the pathway of LPS transport through
LptDE, showing that LPS accesses the periplasmic N-terminal
domain, the hole between the first and last b-strands, the luminal gate, the lumen of the LptD b-barrel, and extracellular
loops 1 and 4 [40].
One important unanswered question for the Lpt system is
the following: how is the transport of the O-antigen portion
of LPS coordinated through the periplasm? LPS can have an
O-antigen region composed of up to 200 sugar rings [11].
This polysaccharide chain can be of a considerable volume
that must be translocated through the periplasm and an LptD
lumen having dimensions of around 35 45 Å. The peptidoglycan in the periplasm also forms a mesh-like barrier that
would not allow easy passage of such a large sugar chain. In
a current model [10], the LPS molecules are lined up like PEZ
candy in the LptC-LptA-LptD trans-periplasmic bridge that
resembles a PEZ dispenser (electronic supplementary material,
figure S1). After the topmost LPS molecule is loaded into the
outer leaflet of the outer membrane and its respective
O-antigen completely translocated to the extracellular space, a
new LPS molecule can be loaded on the inner membrane side
of the bridge. This model works best for LPS molecules that
have very short or no O-antigens. However, bulkier O-antigen
head groups might limit the close alignment of the Lipid A moieties from LPS in the trans-periplasmic bridge, suggesting that
the translocation of the O-antigen would constitute the ratelimiting step in LPS translocation to the outer membrane. LptE
may play a role in opening up the b-barrel lumen to facilitate
O-antigen translocation. However, many (most?) of the
molecular details of this system remain to be worked out.
A second important question is how a new class of peptidomimetic antibiotics interacts with LptD and other OMPs.
One such compound was initially thought to specifically interact with the N-terminal domain of Pseudomonas aeruginosa
LptD [41]; however, a recently described peptidomimetic compound targeting E. coli appears to disrupt the outer membrane
by interacting with various OMPs, including LptD and BamA
Phil. Trans. R. Soc. B 372: 20160224
LptDE core
LptDE full complex
rstb.royalsocietypublishing.org
molecule
4
Downloaded from http://rstb.royalsocietypublishing.org/ on July 31, 2017
5. The outer-membrane protein insertion
complex BamABDCE
6. BamA catalyses insertion of nascent outermembrane proteins into the outer membrane
Structures were determined for full-length BamA from
Neisseria gonorrhoeae and an N-terminally truncated BamA
from Haemophilus ducreyi (figure 3) [32]. In these organisms
as well as in E. coli [33,34], the structure consists of five
periplasmic polypeptide translocation-associated (POTRA)
domains followed by a membrane-inserted b-barrel with 16
antiparallel b-strands. Studies of POTRA-barrel chimeras
have demonstrated that the b-barrel domain is often interchangeable, while the POTRA domains show more species
specificity [48]. The b-barrel is a very unusual OMP, displaying a reduced hydrophobic thickness where strands b1 and
b16 are joined compared with the opposite side of the
b-barrel. This feature was observed by using the crystal structures to measure the distances between aromatic residues on
the outside of the barrel [32], by molecular dynamics simulations that showed a reduced distance between lipid head
groups and a corresponding increased disorder of surrounding lipid chains [32], and by direct observation using electron
microscopy of lipid vesicles with BamA inserted [49]. The
hydrophobic thickness mismatch between BamA and
the outer membrane appears to locally thin and disorder
b16
b1
b1
Neisseria
Haemophilus
P5
P5
P2
P3
P4
P4
P1
Figure 3. Structure of Neisseria gonorrhoeae and Haemophilus ducreyi BamA.
The Neisseria structure has five POTRA domains (P1 to P5), while in the Haemophilus only two are visible. The different number of hydrogen bonds (yellow
dashed lines) between strands b1 and b16 (red) are shown in the insets.
the lipid bilayer, creating a region of the bilayer where
newly folded OMPs may be inserted more efficiently.
In vitro folding experiments show that BamA catalyses the
folding/insertion of OMPs by lowering the energetic barrier
imposed by phosphatydylethanolamine lipids found in the
inner leaflet of the outer membrane [50]. BamA presumably
accomplishes this feat by creating bilayer defects.
Another unusual property of the BamA b-barrel may also
influence the folding and/or insertion of nascent OMPs.
When the structure of N. gonorrhoeae BamA was determined,
it showed that strands b1 and b16 were connected by only
two hydrogen bonds near the extracellular surface [32], in contrast with other pairs of strands in the b-barrel that are
generally linked by eight or more hydrogen bonds (inset,
figure 3). In fact, most of strand b16 adopts a random coil secondary structure and is tucked inside the b-barrel lumen.
Molecular dynamics simulations analysing the stability of the
b-barrel demonstrated that strands b1 and b16 separate completely during a 1 ms simulation; they also re-associate
during the same time period. Simulations were performed at
340 K or 310 K using a symmetric dimyristoyl-phosphatydylethanolamine (DMPE) lipid bilayer; under these conditions
the LptDE b-barrel does not open, suggesting greater
b-barrel instability, or propensity for b-strand separation, for
BamA. Biochemical experiments subsequently confirmed
that this lateral opening event is required for BamA function
by engineering pairs of cysteine residues to form a disulfide
bond at the extracellular surface, in the middle of the bilayer,
or at the periplasmic surface. In all cases, tethering BamA
closed abolished function, and reducing the disulfide to release
the tether restored activity [51]. The lateral opening between
strands b1 and b16 exposes the edges of those b-strands,
which is energetically unfavourable and may allow for
5
Phil. Trans. R. Soc. B 372: 20160224
Just as LPS is synthesized in the cytoplasm and transported
across the entire cell envelope to reach the outer membrane,
proteins targeted to the outer membrane make a similar journey. Proteins residing in the outer membrane adopt a b-barrel
fold, with antiparallel b-strands connected to one another
through extensive hydrogen bonding, where the first and
last b-strands connect to form a b-barrel with a hydrophobic
exterior and hydrophilic lumen. The individual strands of
the b-barrel are amphipathic, with one side hydrophilic and
the other side hydrophobic. Only when the b-barrel is fully
formed, is a continuous hydrophobic surface presented to
the lipid bilayer. This architecture presents complications for
folding of nascent OMPs prior to insertion into the membrane.
Proteins destined for the outer membrane are synthesized
in the cytoplasm and secreted across the inner membrane in an
unfolded state by the Sec translocon [43]. In the periplasm, the
nascent OMP interacts with chaperones such as SurA, Skp,
FkpA and the bifunctional protease/chaperone DegP
[44 –46]. These protein –chaperone complexes are then targeted to the b-barrel assembly machinery (BAM) complex
(figure 1). In E. coli, the BAM complex consists of BamA, an
integral outer membrane protein with an extended periplasmic domain, and BamB, BamC, BamD and BamE, all
lipoproteins anchored to the inner leaflet of the outer membrane [35,47]. The BAM complex is thought to help fold and
insert the nascent protein into the outer membrane, but details
of the mechanism are still being worked out. In the following
sections, we discuss what is currently understood from a
structural perspective and which issues remain to be resolved.
b16
rstb.royalsocietypublishing.org
[42]. The molecular mechanism by which these antibiotics
interact with OMPs to disrupt the outer membrane remains
to be established.
Downloaded from http://rstb.royalsocietypublishing.org/ on July 31, 2017
6
extracellular
outer membrane
P5
BamD
BamC
P2
BamE
P1
BamB
Figure 4. Structure of the BAM complex. This view was generated by merging the crystal structures of BamACDE (PDB code 5EKQ) and BamAB (4PKI). BamA is
shown in cyan, the POTRA domains in different shades of teal, BamB in grey, BamC in green, BamD in gold and BamE in violet. The first and last b-strands of the
BamA barrel are highlighted in red.
b-augmentation, or strand pairing, with other b-strands, such
as those of the nascent OMP. This could potentially allow for
the coordinated folding and insertion of a nascent OMP by
directly budding it off from the BamA opening [32]. The opening also creates a pathway from the lumen of the b-barrel
directly into the lipid bilayer, although exactly what this interface looks like is currently unknown. Located just above the
barrel opening is an ‘exit pore’ defined primarily by alternating
movements of extracellular loop 1 [51]. The exit pore may facilitate transport of extracellular loops (which can be as long as 100
residues [52]) of the nascent OMP to the cell surface. These features have been found in all BamA structures solved to date
(table 1), and may play a role in folding nascent OMPs or
inserting them into the outer membrane, or perhaps both.
7. Structures of the BAM complex reveal further
BamA movements
By 2011, the structures of all of the lipoproteins, BamB, BamC,
BamD and BamE, had been determined and a model for assembly of the BAM complex was formulated (for a review, see [53]).
Recent structures of BamB and BamD bound to a periplasmic
fragment of BamA, as well as an earlier structure of a BamCD
complex, confirmed the model and helped to visualize component assembly [54–56]. It has become clear that the BamA
POTRA domains can exhibit flexibility in the absence of other
BAM components [57–59] but they probably interact with the
periplasmic surface of the outer membrane in vivo, modulating
their flexibility [60]. BamD plays an important role in outermembrane protein biogenesis [61,62], while the trapping of a
folding intermediate suggested that BamA and BamD work
together to catalyse folding [63].
Although the model correctly predicted many of the interactions made by individual lipoproteins with the BamA
POTRA domains, and the recent structural and functional
studies illustrated a number of important features, we could
not have anticipated the unusual conformational changes in
BamA occurring upon BAM complex formation.
In 2010, an intact BAM complex was prepared by making
sub-complexes containing BamAB and BamCDE, and then
reconstituting the BAM complex into lipid vesicles. Upon
addition of the chaperone SurA and an unfolded OMP substrate
(OmpT), the BAM complex was able to fold and insert the OMP
substrate into vesicles without any additional protein components or energy requirement [64]. This in vitro system was
recently improved upon by expressing all five Bam components
from a single expression vector, resulting in a BAM complex
with markedly increased folding activity [65]. The improved
method for producing an intact BAM complex assisted three
groups in determining crystal structures of E. coli BamACDE
and BamABCDE (table 1 and figure 4) [29–31].
Purification of the BAM complex resulted in stoichiometric amounts of BamA, BamB, BamC, BamD and BamE
after size exclusion chromatography [29], but BamB dissociates or is degraded during crystallization. Of the four
Phil. Trans. R. Soc. B 372: 20160224
periplasm
rstb.royalsocietypublishing.org
BamA
Downloaded from http://rstb.royalsocietypublishing.org/ on July 31, 2017
b16
b1
recently published BAM complex structures, two contain
BamB [30,31] and two do not [29,30]. The Bam lipoproteins
assemble on the BamA POTRA domains essentially as predicted from earlier structural and functional/genetics
studies (reviewed in [53]), with BamB on one side primarily
interacting with POTRA 3 and BamCDE on the other side,
where BamD interacts primarily with POTRA 5 (with a smaller number of interactions with POTRA 2) and BamE also
interacts with POTRA 5. BamC binds to BamD and does
not make direct contact with the BamA POTRA domains in
three of the four structures. However, in one of the structures,
the C-terminal domain of BamC interacts with POTRA 2 [30].
In the two BamABCDE structures, the BamA b-barrel is
fully closed, and POTRA 5 is rotated about 308 from the
b-barrel domain, allowing access to the b-barrel lumen. In
the two BamACDE structures, POTRA 5 is located directly
beneath the b-barrel lumen, excluding access, and the barrel
itself is more open than was observed in the structure of
BamA alone (figure 5). Binding of BamCDE to BamA induces
a large conformational twist in the BamA b-barrel, where the
first eight b-strands rotate away from strand b16. The most
dramatic change is observed in strand b1, which shows a
rotation of about 458. The resulting BamA b-barrel makes
no hydrogen bonds at all between strands b1 and b16, with
only a single hydrogen bond linking the beginning of
strand b1 to the periplasmic loop connecting strands b14
8. Conclusion
We have reviewed two major players of outer-membrane biogenesis: the LPS and the bacterial assembly machineries. Both
complexes are essential for the integrity and survival of
Gram-negative bacteria, and while both translocate basic
building blocks of the bacterial outer membrane, their mechanism of action is markedly different. A series of crystal
structures complemented by mutagenesis and molecular
dynamics simulations offer a glimpse into the intricate mechanisms of these highly complex molecular machines. Further
experiments are needed to fully understand the details of
these processes and permit the design of molecules that
target specific parts of the BAM and Lpt pathways, with
potential therapeutic benefits.
Note added in proof
A recent cryo-EM structure of the BAM complex contains all
five Bam proteins and shows a closed BamA barrel (Iadanza
et al., Nat. Comm. 7:12865 (2016).
Authors’ contributions. I.B., N.N. and S.K.B. wrote the manuscript and
approved the final version.
Competing interests. We declare we have no competing interests.
Funding. I.B. and S.K.B. are supported by the Intramural Research
Program of the National Institutes of Health, National Institute of
Diabetes and Digestive and Kidney Diseases (ZIA DK036139-10
LMB). N.N. is supported by the Department of Biological Sciences
at Purdue University and by the National Institute of Allergy and
Infectious Diseases (1K22AI113078-01).
References
1.
Beveridge TJ. 1999 Structures of gram-negative cell
walls and their derived membrane vesicles.
J. Bacteriol. 181, 4725– 4733.
2.
Bishop RE. 2014 Structural biology:
Lipopolysaccharide rolls out the barrel.
Nature 511, 37 –38. (doi:10.1038/nature13508)
3.
Li L, Park E, Ling J, Ingram J, Ploegh H,
Rapoport TA. 2016 Crystal structure of a
substrate-engaged SecY protein-translocation
7
Phil. Trans. R. Soc. B 372: 20160224
Figure 5. Conformational changes in the BamA b-barrel and POTRA 5. A
rotational movement of the first eight strands of the b-barrel and a translational movement of POTRA 5 are observed by comparing the BamACDE
structure (open barrel, PDB code 5EKQ) with the BamABCDE structure
(closed barrel, PDB code 5D0O).
and b15. The movement changes the angle of the affected
b-strands in the membrane, opens the exit pore through conformational changes in extracellular loops 1–3, and causes a
large rearrangement of the b-barrel opening to the lipid
bilayer (electronic supplementary material, movie S1).
Whether BamB is actively modulating these conformational
changes, and exactly how these movements assist folding
and insertion of nascent OMPs, is unknown.
There are many questions remaining in the BAM field, and
the structures of Bam components and BAM complexes provide a framework for future experiments. Among the
unresolved issues are the following: how folded or unfolded
is the substrate OMP when bound to chaperones? How many
copies of a chaperone bind to a single OMP? As different
classes of OMPs display dependencies on different chaperones
[45], does that indicate that there are multiple folding/insertion
mechanisms facilitated by the BAM complex? What does the
interaction between a chaperone –OMP complex and the
BAM complex look like? What exactly is the BAM complex
doing: does it actively fold OMPs, or simply facilitate insertion
by local destabilization of the outer membrane, or does it participate in both folding and insertion? How similar is
biogenesis of OMPs in mitochondria and chloroplasts [66,67]?
rstb.royalsocietypublishing.org
5EKQ
5D0O
Downloaded from http://rstb.royalsocietypublishing.org/ on July 31, 2017
5.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31. Han L et al. 2016 Structure of the BAM complex
and its implications for biogenesis of outermembrane proteins. Nat. Struct. Mol. Biol. 23,
192–196. (doi:10.1038/nsmb.3181)
32. Noinaj N, Kuszak AJ, Gumbart JC, Lukacik P, Chang
H, Easley NC, Lithgow T, Buchanan SK. 2013
Structural insight into the biogenesis of beta-barrel
membrane proteins. Nature 501, 385–390. (doi:10.
1038/nature12521)
33. Albrecht R, Schutz M, Oberhettinger P, Faulstich M,
Bermejo I, Rudel T, Diederichs K, Zeth K. 2014
Structure of BamA, an essential factor in outer
membrane protein biogenesis. Acta Crystallogr. D
Biol. Crystallogr. 70, 1779– 1789. (doi:10.1107/
S1399004714007482)
34. Ni D et al. 2014 Structural and functional analysis of
the beta-barrel domain of BamA from Escherichia
coli. FASEB J. 28, 2677 –2685. (doi:10.1096/fj.13248450)
35. Wu T, Malinverni J, Ruiz N, Kim S, Silhavy TJ, Kahne
D. 2005 Identification of a multicomponent complex
required for outer membrane biogenesis in
Escherichia coli. Cell 121, 235–245. (doi:10.1016/j.
cell.2005.02.015)
36. Chimalakonda G, Ruiz N, Chng SS, Garner RA, Kahne
D, Silhavy TJ. 2011 Lipoprotein LptE is required for
the assembly of LptD by the beta-barrel assembly
machine in the outer membrane of Escherichia coli.
Proc. Natl Acad. Sci. USA 108, 2492–2497. (doi:10.
1073/pnas.1019089108)
37. Freinkman E, Okuda S, Ruiz N, Kahne D. 2012
Regulated assembly of the transenvelope protein
complex required for lipopolysaccharide export.
Biochemistry 51, 4800 –4806. (doi:10.1021/
bi300592c)
38. Grabowicz M, Yeh J, Silhavy TJ. 2013 Dominant
negative lptE mutation that supports a role for LptE
as a plug in the LptD barrel. J. Bacteriol. 195,
1327– 1334. (doi:10.1128/JB.02142-12)
39. van den Berg B, Black PN, Clemons Jr WM,
Rapoport TA. 2004 Crystal structure of the longchain fatty acid transporter FadL. Science 304,
1506– 1509. (doi:10.1126/science.1097524)
40. Li X, Gu Y, Dong H, Wang W, Dong C. 2015 Trapped
lipopolysaccharide and LptD intermediates reveal
lipopolysaccharide translocation steps across the
Escherichia coli outer membrane. Sci. Rep. 5, 11883.
(doi:10.1038/srep11883)
41. Srinivas N et al. 2010 Peptidomimetic antibiotics
target outer-membrane biogenesis in Pseudomonas
aeruginosa. Science 327, 1010–1013. (doi:10.1126/
science.1182749)
42. Urfer M et al. 2016 A peptidomimetic antibiotic
targets outer membrane proteins and disrupts
selectively the outer membrane in Escherichia coli.
J. Biol. Chem. 291, 1921 –1932. (doi:10.1074/jbc.
M115.691725)
43. Pugsley AP. 1993 The complete general secretory
pathway in gram-negative bacteria. Microbiol. Rev.
57, 50 – 108.
44. Costello SM, Plummer AM, Fleming PJ, Fleming KG.
2016 Dynamic periplasmic chaperone reservoir
facilitates biogenesis of outer membrane proteins.
8
Phil. Trans. R. Soc. B 372: 20160224
6.
17.
across the periplasm in E. coli. Science 338, 1214–
1217. (doi:10.1126/science.1228984)
Sherman DJ, Lazarus MB, Murphy L, Liu C, Walker S,
Ruiz N, Kahne D. 2014 Decoupling catalytic activity
from biological function of the ATPase that powers
lipopolysaccharide transport. Proc. Natl Acad. Sci. USA
111, 4982 –4987. (doi:10.1073/pnas.1323516111)
Chng SS, Ruiz N, Chimalakonda G, Silhavy TJ, Kahne
D. 2010 Characterization of the two-protein complex
in Escherichia coli responsible for lipopolysaccharide
assembly at the outer membrane. Proc. Natl Acad.
Sci. USA 107, 5363 –5368. (doi:10.1073/pnas.
0912872107)
Botos I, Majdalani N, Mayclin SJ, McCarthy JG,
Lundquist K, Wojtowicz D, Barnard TJ, Gumbart JC,
Buchanan SK. 2016 Structural and functional
characterization of the LPS transporter LptDE from
Gram-negative pathogens. Structure 24, 965–976.
(doi:10.1016/j.str.2016.03.026)
Qiao S, Luo Q, Zhao Y, Zhang XC, Huang Y. 2014
Structural basis for lipopolysaccharide insertion
in the bacterial outer membrane. Nature 511,
108 –111. (doi:10.1038/nature13484)
Dong H et al. 2014 Structural basis for outer
membrane lipopolysaccharide insertion. Nature 511,
52 –56. (doi:10.1038/nature13464)
Malojcic G, Garner RA, Kahne D. 2014 Crystal
structure of the lipopolysaccharide assembly
complex LptD-LptE from the Escherichia coli outer
membrane. PDB code 4RHB.
Genomics JCfS. 2013 Crystal structure of a putative
lipoprotein (CC_3750) from Caulobacter crescentus
CB15 at 2.40 A resolution. PDB code 4KWY.
Vorobiev SM et al. 2007 Crystal structure of the
A1KSW9_NEIMF protein from Neisseria
meningitidis. Northeast Structural Genomics
Consortium target MR36a. PDB code 3BF2.
Malojcic G, Andres D, Grabowicz M, George AH, Ruiz
N, Silhavy TJ, Kahne D. 2014 LptE binds to and
alters the physical state of LPS to catalyze its
assembly at the cell surface. Proc. Natl Acad. Sci.
USA 111, 9467–9472. (doi:10.1073/pnas.
1402746111)
Forouhar F et al. 2007 Crystal structure of the rare
lipoprotein B (SO_1173) from Shewanella
oneidensis, Northeast Structural Genomics
Consortium Target SoR91A. PDB code 2R76.
Rossi P et al. 2007 Solution NMR structure of
uncharacterized lipoprotein B from Nitrosomonas
europaea. Northeast Structural Genomics target
NeR45A. PDB code 2JXP.
Moehle K, Kocherla H, Bacsa B, Jurt S, Zerbe K,
Robinson JA, Zerbe O. 2016 Solution structure and
dynamics of LptE from Pseudomonas aeruginosa.
Biochemistry 55, 2936 –2943. (doi:10.1021/acs.
biochem.6b00313)
Bakelar J, Buchanan SK, Noinaj N. 2016 The
structure of the beta-barrel assembly machinery
complex. Science 351, 180 –186. (doi:10.1126/
science.aad3460)
Gu Y et al. 2016 Structural basis of outer membrane
protein insertion by the BAM complex. Nature 531,
64 –69. (doi:10.1038/nature17199)
rstb.royalsocietypublishing.org
4.
channel. Nature 531, 395–399. (doi:10.1038/
nature17163)
Park E, Menetret JF, Gumbart JC, Ludtke SJ, Li W,
Whynot A, Rapoport TA, Akey CW. 2014 Structure of
the SecY channel during initiation of protein
translocation. Nature 506, 102 –106. (doi:10.1038/
nature12720)
Hearn EM, Patel DR, Lepore BW, Indic M, van den Berg
B. 2009 Transmembrane passage of hydrophobic
compounds through a protein channel wall. Nature
458, 367–370. (doi:10.1038/nature07678)
van den Berg B. 2010 Going forward laterally:
transmembrane passage of hydrophobic molecules
through protein channel walls. ChemBioChem 11,
1339–1343. (doi:10.1002/cbic.201000105)
van den Berg B. 2010 Bacterial cleanup: lateral
diffusion of hydrophobic molecules through protein
channel walls. Biomol. Concepts 1, 263–270.
(doi:10.1515/bmc.2010.024)
Ahn VE, Lo EI, Engel CK, Chen L, Hwang PM, Kay LE,
Bishop RE, Prive GG. 2004 A hydrocarbon ruler
measures palmitate in the enzymatic acylation of
endotoxin. EMBO J. 23, 2931– 2941. (doi:10.1038/
sj.emboj.7600320)
Khan MA, Bishop RE. 2009 Molecular mechanism
for lateral lipid diffusion between the outer
membrane external leaflet and a beta-barrel
hydrocarbon ruler. Biochemistry 48, 9745 –9756.
(doi:10.1021/bi9013566)
Okuda S, Sherman DJ, Silhavy TJ, Ruiz N, Kahne D.
2016 Lipopolysaccharide transport and assembly at
the outer membrane: the PEZ model. Nat. Rev.
Microbiol. 14, 337–345. (doi:10.1038/nrmicro.
2016.25)
Whitfield C, Trent MS. 2014 Biosynthesis and export
of bacterial lipopolysaccharides. Annu. Rev. Biochem.
83, 99 –128. (doi:10.1146/annurev-biochem060713-035600)
Villa R et al. 2013 The Escherichia coli Lpt
transenvelope protein complex for
lipopolysaccharide export is assembled via
conserved structurally homologous domains.
J. Bacteriol. 195, 1100– 1108. (doi:10.1128/JB.
02057-12)
Narita S, Tokuda H. 2009 Biochemical
characterization of an ABC transporter LptBFGC
complex required for the outer membrane sorting
of lipopolysaccharides. FEBS Lett. 583, 2160– 2164.
(doi:10.1016/j.febslet.2009.05.051)
Sperandeo P, Villa R, Martorana AM, Samalikova M,
Grandori R, Deho G, Polissi A. 2011 New insights
into the Lpt machinery for lipopolysaccharide
transport to the cell surface: LptA-LptC interaction
and LptA stability as sensors of a properly
assembled transenvelope complex. J. Bacteriol. 193,
1042–1053. (doi:10.1128/JB.01037-10)
Bowyer A, Baardsnes J, Ajamian E, Zhang L, Cygler
M. 2011 Characterization of interactions between
LPS transport proteins of the Lpt system. Biochem.
Biophys. Res. Commun. 404, 1093–1098. (doi:10.
1016/j.bbrc.2010.12.121)
Okuda S, Freinkman E, Kahne D. 2012 Cytoplasmic
ATP hydrolysis powers transport of lipopolysaccharide
Downloaded from http://rstb.royalsocietypublishing.org/ on July 31, 2017
46.
47.
49.
50.
51.
52.
60. Fleming PJ, Patel DS, Wu EL, Qi Y, Yeom MS, Sousa
MC, Fleming KG, Im W. 2016 BamA POTRA domain
interacts with a native lipid membrane surface.
Biophys. J. 110, 2698– 2709. (doi:10.1016/j.bpj.
2016.05.010)
61. Hagan CL, Wzorek JS, Kahne D. 2015 Inhibition of
the beta-barrel assembly machine by a peptide that
binds BamD. Proc. Natl Acad. Sci. USA 112, 2011–
2016. (doi:10.1073/pnas.1415955112)
62. Misra R, Stikeleather R, Gabriele R. 2015 In vivo
roles of BamA, BamB and BamD in the biogenesis
of BamA, a core protein of the beta-barrel assembly
machine of Escherichia coli. J. Mol. Biol. 427,
1061– 1074. (doi:10.1016/j.jmb.2014.04.021)
63. Lee J et al. 2016 Characterization of a stalled
complex on the beta-barrel assembly machine. Proc.
Natl Acad. Sci. USA 113, 8717–8722. (doi:10.1073/
pnas.1604100113)
64. Hagan CL, Kim S, Kahne D. 2010 Reconstitution of
outer membrane protein assembly from purified
components. Science 328, 890–892. (doi:10.1126/
science.1188919)
65. Roman-Hernandez G, Peterson JH, Bernstein HD.
2014 Reconstitution of bacterial autotransporter
assembly using purified components. Elife 3,
e04234. (doi:10.7554/eLife.04234)
66. Ulrich T, Rapaport D. 2015 Biogenesis of beta-barrel
proteins in evolutionary context. Int. J. Med.
Microbiol. 305, 259– 264. (doi:10.1016/j.ijmm.
2014.12.009)
67. Hohr AI, Straub SP, Warscheid B, Becker T,
Wiedemann N. 2015 Assembly of beta-barrel
proteins in the mitochondrial outer membrane.
Biochim. Biophys. Acta 1853, 74– 88. (doi:10.1016/
j.bbamcr.2014.10.006)
9
Phil. Trans. R. Soc. B 372: 20160224
48.
53. O’Neil PK, Rollauer SE, Noinaj N, Buchanan SK. 2015
Fitting the pieces of the beta-barrel assembly
machinery complex. Biochemistry 54, 6303–6311.
(doi:10.1021/acs.biochem.5b00852)
54. Bergal HT, Hopkins AH, Metzner SI, Sousa MC. 2016
The structure of a BamA-BamD fusion illuminates
the architecture of the beta-barrel assembly
machine core. Structure 24, 243 –251. (doi:10.1016/
j.str.2015.10.030)
55. Kim KH, Aulakh S, Paetzel M. 2011 Crystal structure
of beta-barrel assembly machinery BamCD protein
complex. J. Biol. Chem. 286, 39 116–39 121.
(doi:10.1074/jbc.M111.298166)
56. Jansen KB, Baker SL, Sousa MC. 2015 Crystal
structure of BamB bound to a periplasmic
domain fragment of BamA, the central
component of the beta-barrel assembly machine.
J. Biol. Chem. 290, 2126–2136. (doi:10.1074/jbc.
M114.584524)
57. Morgado L, Zeth K, Burmann BM, Maier T, Hiller S.
2015 Characterization of the insertase BamA in
three different membrane mimetics by solution
NMR spectroscopy. J. Biomol. NMR 61, 333– 345.
(doi:10.1007/s10858-015-9906-y)
58. Sinnige T, Weingarth M, Daniels M, Boelens R, Bonvin
AM, Houben K, Baldus M. 2015 Conformational
plasticity of the POTRA 5 domain in the outer
membrane protein assembly factor BamA. Structure
23, 1317 –1324. (doi:10.1016/j.str.2015.04.014)
59. Sinnige T, Houben K, Pritisanac I, Renault M,
Boelens R, Baldus M. 2015 Insight into the
conformational stability of membrane-embedded
BamA using a combined solution and solid-state
NMR approach. J. Biomol. NMR 61, 321–332.
(doi:10.1007/s10858-014-9891-6)
rstb.royalsocietypublishing.org
45.
Proc. Natl Acad. Sci. USA 113, E4794 –E4800.
(doi:10.1073/pnas.1601002113)
Mahoney TF, Ricci DP, Silhavy TJ. 2016 Classifying
beta-barrel assembly substrates by manipulating
essential bam complex members. J. Bacteriol. 198,
1984–1992. (doi:10.1128/JB.00263-16)
Plummer AM, Fleming KG. 2016 From chaperones
to the membrane with a BAM! Trends Biochem. Sci.
41, 872–882. (doi:10.1016/j.tibs.2016.06.005)
Buchanan SK, Noinaj N. 2015 The BAM complex
(ed. JM Walker). New York, NY: Humana Press.
Browning DF et al. 2015 Cross-species chimeras
reveal BamA POTRA and beta-barrel domains must
be fine-tuned for efficient OMP insertion. Mol.
Microbiol. 97, 646 –659. (doi:10.1111/mmi.13052)
Sinnige T, Weingarth M, Renault M, Baker L,
Tommassen J, Baldus M. 2014 Solid-state NMR
studies of full-length BamA in lipid bilayers
suggest limited overall POTRA mobility. J. Mol.
Biol. 426, 2009–2021. (doi:10.1016/j.jmb.2014.
02.007)
Gessmann D, Chung YH, Danoff EJ, Plummer AM,
Sandlin CW, Zaccai NR, Fleming KG. 2014 Outer
membrane beta-barrel protein folding is physically
controlled by periplasmic lipid head groups and
BamA. Proc. Natl Acad. Sci. USA 111, 5878 –5883.
(doi:10.1073/pnas.1322473111)
Noinaj N, Kuszak AJ, Balusek C, Gumbart JC,
Buchanan SK. 2014 Lateral opening and exit
pore formation are required for BamA function.
Structure 22, 1055 –1062. (doi:10.1016/j.str.2014.
05.008)
Noinaj N et al. 2012 Structural basis for iron piracy
by pathogenic Neisseria. Nature 483, 53 –58.
(doi:10.1038/nature10823)