Mechanistic link between β barrel assembly and
the initiation of autotransporter secretion
Olga Pavlova, Janine H. Peterson, Raffaele Ieva1, and Harris D. Bernstein2
Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
Edited by Linda L. Randall, University of Missouri, Columbia, MO, and approved January 24, 2013 (received for review November 1, 2012)
Autotransporters are bacterial virulence factors that contain an
N-terminal extracellular (“passenger”) domain and a C-terminal
β barrel (“β”) domain that anchors the protein to the outer mem-
brane. The β domain is required for passenger domain secretion,
but its exact role in autotransporter biogenesis is unclear. Here we
describe insights into the function of the β domain that emerged
from an analysis of mutations in the Escherichia coli O157:H7 auto-
transporter EspP. We found that the G1066A and G1081D muta-
tions slightly distort the structure of the β domain and delay the
initiation of passenger domain translocation. Site-specific photo-
crosslinking experiments revealed that the mutations slow the in-
sertion of the β domain into the outer membrane, but do not delay
the binding of the β domain to the factor that mediates the in-
sertion reaction (the Bam complex). Our results demonstrate that
the β domain does not simply target the passenger domain to the
outer membrane, but promotes translocation when it reaches
a specific stage of assembly. Furthermore, our results provide ev-
idence that the Bam complex catalyzes the membrane integration
of β barrel proteins in a multistep process that can be perturbed by
minor structural defects in client proteins.
membrane protein assembly|outer membrane proteins|
consist of an N-terminal extracellular domain (passenger do-
main) and a C-terminal β barrel domain (β domain) that anchors
the protein to the outer membrane (OM) (1). Passenger domains
range in size from ∼20 kDa to over 400 kDa and have been
shown to mediate a variety of different virulence functions (2).
Following their translocation across the OM, many passenger
domains are released from the cell surface by a proteolytic
cleavage. Experimental and in silico studies have suggested that
virtually all passenger domains form a β-helical structure, despite
the fact that their primary amino acid sequence is poorly con-
served (3–6). β domains are generally ∼30 kDa in size, and al-
though they also display considerable sequence diversity, they
can all be identified as members of the pfam03797 (smart00869)
family of protein domains. Several divergent β domains have
been crystallized and have been shown to form nearly superim-
posable 12-stranded β barrels that are traversed by an α-helical
segment (7–10). The α-helical segment generally extends into the
extracellular space and links the passenger domain to the β do-
main. In a few cases, however, the passenger domain is released
in an intrabarrel cleavage reaction that leaves a small α-helical
segment inside the barrel (11). Available evidence suggests that
the incorporation of the α-helical segment into the β domain
pore occurs in the periplasm (where the β domain appears to
undergo considerable folding) and is required for the integration
of the β domain into the OM (12).
Although there is general agreement that the passenger do-
main is translocated across the OM in a C- to N-terminal di-
rection (13, 14), the mechanism of translocation has been hotly
debated. Early experiments in which the β domain was deleted
showed that it plays an essential role in translocation and led to
the proposal that it forms a channel through which the covalently
linked passenger domain is secreted (15). Recently, however,
several findings have challenged the “autotransporter” hypoth-
utotransporters are a very large superfamily of virulence
factors produced by Proteobacteria and Chlamydia that
esis. Crystallographic analysis has shown that the pore formed
by the β domain is ∼10 Å in diameter and therefore only wide
enough to accommodate a completely unfolded polypeptide in
a hairpin conformation or a single polypeptide in an α-helical
conformation. Molecular dynamics simulations have confirmed
that the β domain is extremely stable and unlikely to expand
spontaneously (16, 17). Nevertheless, both native and modified
passenger domains that have acquired tertiary structure in the
periplasm are secreted efficiently by the autotransporter pathway
(18, 19). Furthermore, the observation that the peptide inside
the β domain is in an α-helical conformation at an early stage of
translocation is incompatible with passenger domain trans-
location through the β domain pore (20). Finally, crosslinking
experiments (13) have shown that during its transit across the
OM, the passenger domain interacts with BamA, a component
of a complex that binds to β barrel proteins and facilitates their
integration into the OM by an unknown mechanism (21–24).
Interestingly, members of the BamA superfamily produced by
bacteria and chloroplasts have been shown to mediate protein
translocation reactions (25). In addition to BamA, which consists
of a β barrel domain and five periplasmic POTRA (polypeptide
transport associated) domains, the Bam complex contains four
lipoproteins called BamB, BamC, BamD, and BamE.
An analysis of the interactions between cellular factors and the
β domain ofa model autotransporter produced by Escherichia coli
O157:H7calledEspP hasrecentlyledtoanew modelinwhich the
translocation of the passenger domain and the assembly of the β
domain are interconnected (26). EspP is a member of the SPATE
(serine protease autotransporters of Enterobacteraceae) family of
autotransporters whose passenger domains are released in an
intrabarrel cleavage reaction (11). Site-specific photocrosslinking
experiments showed that the EspP β domain interacts with the
periplasmic chaperone Skp and components of the Bam complex
in a temporally and spatially regulated fashion in vivo. Skp is
Most proteins that reside in the bacterial outer membrane are β
sheets that fold into a unique cylindrical structure known as
a “β barrel.” Here we describe significant insights into the
function of the Bam complex, a protein machine that catalyzes
the insertion of β barrel proteins into the membrane by an
unknown mechanism. By analyzing the assembly of auto-
transporters, a specialized family of outer membrane proteins,
we found that the function of the Bam complex can be divided
into an initial substrate binding stage and a subsequent in-
sertion stage that is surprisingly sensitive to structural dis-
tortions in client proteins.
Author contributions: O.P., R.I., and H.D.B. designed research; O.P., J.H.P., R.I., and H.D.B.
performed research; O.P., R.I., and H.D.B. analyzed data; and O.P. and H.D.B. wrote
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1Present address: Institute for Biochemistry and Molecular Biology, University of Freiburg,
D-79104 Freiburg, Germany.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| Published online February 19, 2013www.pnas.org/cgi/doi/10.1073/pnas.1219076110
a homotrimer that resembles a jellyfish with long, flexible α-helical
tentacles that form a large central cavity (27, 28). The results
revealed that although the entire β domain initially interacts with
Skp, discrete regions of the polypeptide subsequently interact with
BamA, BamB, and BamD. The data suggest the existence of an
assembly intermediate in which the EspP β domain is effectively
surrounded by components of the Bam complex. Interestingly, the
results also suggested that the passenger domain is not only nor-
mally secreted and cleaved before the completion of β domain
assembly, but that the completion of β domain assembly is strictly
dependent on the completion of passenger domain translocation.
To account for these results and other recent observations on
autotransporter biogenesis, it was proposed that the passenger
domain is secreted through a channel comprised of an incom-
pletely closed β domain, BamA, and possibly other factors that
have not yet been identified (26).
Although these results provide insight into the later stages of
autotransporter assembly, they do not address the mechanism by
which the translocation of the passenger domain across the OM
is initiated. One possibility is that once the β domain captures the
appropriate α-helical peptide in the periplasm, it simply serves as
a targeting signal that guides the passenger domain to the OM.
In that case, it is likely that the initiation of passenger domain
translocation would be closely coupled to the binding of the β
domain to the Bam complex. Alternatively, the initiation of
translocation might depend on the completion of an additional
step in β domain assembly that occurs after the β domain docks
onto the Bam complex. Here we describe an analysis of several
EspP β domain mutants that strongly supports the latter hy-
pothesis. We found that the mutation of two highly conserved
residues, G1066 and G1081, perturbs the stability of the β do-
main and delays the initiation of passenger domain translocation.
In vivo site-specific photocrosslinking and other experiments
showed that the mutations delay both the exposure of the pas-
senger domain on the cell surface after the β domain binds to the
Bam complex and the integration of the β domain into the lipid
bilayer. By uncoupling the initiation of translocation from the
interaction of the β domain with the Bam complex, our results
imply that the β domain must undergo a transition after it rea-
ches the OM before translocation can begin. Moreover, our
results suggest that the Bam complex facilitates the integration
of β barrel proteins into the OM in a reaction that can be divided
into discrete stages.
Mutation of Two Conserved Residues in the EspP β Domain Delays the
Initiation of Passenger Domain Translocation. Although autotrans-
porter β domains do not contain any invariant residues, a multi-
ple sequence alignment of 100 divergent β domains revealed
the presence of 10 highly conserved residues, three of which are
glycines (Fig. S1). Two of the conserved glycine residues (G1066
and G1081) reside in the second and third β strands of the EspP
β domain (residues 1024–1300) (Fig. 1 A and B). Based on the
crystal structure of the EspP β domain (8), the side chain of any
amino acid larger than glycine at position 1066 (including the
methyl group of an alanine residue) is predicted to project into
the lumen of the β barrel and clash sterically with the side chain
of a second highly conserved residue, W1042 (Fig. 1 B and C,
and Fig. S1). Similarly, amino acid side chains at position 1081
are predicted to clash sterically with the side chains of another
highly conserved residue (Y1108) and a moderately conserved
residue (D1068) inside the β barrel (Fig. 1 B and C, and Fig. S1).
We performed a heat modifiability assay to determine the
effect of mutations at positions 1066 and 1081 on the stability of
the EspP β domain. This assay is based on the observation that
native β barrel proteins are often resistant to SDS denaturation
and migrate relatively rapidly on SDS gels unless they are heated
to a high temperature (29). E. coli strain AD202 (MC4100
ompT::kan) transformed with a plasmid encoding wild-type EspP
under the control of the trc promoter (pRLS5) or a derivative of
pRLS5 encoding a mutant EspP protein was grown in minimal
medium. After isopropyl-β-D-thiogalactopyranoside (IPTG) was
added to induce expression of the plasmid-borne gene, cells were
lysed by sonication. Cell extracts were then heated in SDS/PAGE
sample buffer and the cleaved EspP β domain was detected by
Western blot using an antibody generated against a C-terminal
potential steric clashes inside the EspP β barrel. (A)
Illustration of EspP showing the signal peptide (SP)
(residues 1–55), the passenger domain (residues 56–
1023), and the β domain (residues 1024–1300). Pro-
EspP is the precursor form of the protein that is
observed before the proteolytic release of the pas-
senger domain from the β domain. (B) Modeling of
the G1066A and G1081D mutations based on the
crystal structure of the EspP β domain (8). The pas-
senger domain cleavage site is shown. (C) Steric
clashes created by the introduction of the G1066A
(Upper) and G1081D (Lower) mutations were pre-
dicted in Coot using Molprobity tools (41).
Mutation of residues 1066 and 1081 creates
Pavlova et al.PNAS
| Published online February 19, 2013
reached OD550= 0.2, EspP synthesis was induced by the addition of 10 μM
IPTG. After 30 min, cells were collected by centrifugation (2,500 × g, 15 min,
4 °C), resuspended at 10 OD units/mL in PBS, and sonicated. Unbroken cells
were pelleted (2,500 × g, 5 min, 4 °C), and 1 mL of each cell extract was
centrifuged in a Beckman TLA100.2 rotor (100,000 × g, 30 min, 4 °C). In heat
modifiability experiments, the resulting membrane pellet was resuspended
in PBS at 40 OD units/mL. Aliquots were then mixed with 2× Laemmli loading
buffer and heated to a temperature between 25 °C and 90 °C for 15 min.
Proteins were resolved by SDS/PAGE on 8–16% minigels (Life Technologies)
and the EspP β domain was detected by Western blot.
Pulse-Chase Labeling and Photocrosslinking. Cultures were grown as described
above and EspP synthesis was induced by the addition of 10 μM IPTG (for
experiments in which cells contained pRLS5 or pJH61 derivatives) or 200 μM
IPTG (for crosslinking experiments in which cells contained pRl22 deriva-
tives). Pulse-chase labeling and photocrosslinking were performed essen-
tially as previously described (13). In experiments performed at low
temperature, cultures were shifted to 25 °C 7 min before pulse labeling. Cells
were pipetted over ice, concentrated by centrifugation and resuspended in
1 mL M9 medium. Proteins in all samples were collected by TCA precipitation.
In some experiments, resuspended cells were divided in half, and one half
was treated with PK as previously described (13) before TCA precipitation.
Immunoprecipitations were performed as previously described (12), and
proteins were resolved by SDS/PAGE on 8–16% minigels. Percent surface
exposure and percent passenger domain cleavage were calculated as pre-
viously described (20). To detect crosslinking between EspP β′ and LPS, the
radio labeling step was omitted. Cell membranes were isolated as described
above and then resuspended in PBS containing 5% Elugent at 20 OD units/mL
Insoluble material was then removed by centrifugation (TLA100.2 rotor,
100,000 × g, 30 min, 4 °C) before proteins were TCA precipitated and ana-
lyzed by Western blot.
Urea Extractions. Cultures were subjected to pulse-chase labeling essentially
as previously described (13). Cells were then pipetted over ice and collected
by centrifugation. Cell pellets were resuspended in TBS (20 mM Tris pH
7.4/150 mM NaCl) at 3 OD units/mL and sonicated. Unbroken cells were
removed by centrifugation and a portion of the supernatant (representing the
total cell lysate) was removed. The remainder of each sample was centrifuged
in a Beckman TLA100.2 rotor (100,000 × g, 30 min, 4 °C). The resulting su-
pernatant was defined as the soluble fraction and the pellet as the membrane
fraction. The membranes were resuspended in 150 μL 20 mM Tris pH 7.4/100
mM glycine/6M urea and incubated at 25 °C for 1 h (21). The urea insoluble
fraction was then pelleted in a Beckman TLA-100 rotor (150,000 × g, 30 min,
4 °C) and resuspended in TBS. After urea-containing fractions were diluted
1:25 in water, proteins in all samples were collected by TCA precipitation and
resolved by SDS/PAGE on 8–16% minigels.
ACKNOWLEDGMENTS. We thank Travis Barnard for helping to construct Fig.
1 and Yihong Ye for providing valuable comments on the manuscript. This
work was supported by the National Institute of Diabetes and Digestive and
Kidney Diseases Intramural Research Program.
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Pavlova et al.PNAS
| Published online February 19, 2013