Crystal Structures of the Outer Membrane Domain
of Intimin and Invasin from Enterohemorrhagic
E. coli and Enteropathogenic Y. pseudotuberculosis
James W. Fairman,1Nathalie Dautin,3Damian Wojtowicz,2Wei Liu,4Nicholas Noinaj,1Travis J. Barnard,1Eshwar Udho,5
Teresa M. Przytycka,2Vadim Cherezov,4and Susan K. Buchanan1,*
1National Institute of Diabetes and Digestive and Kidney Diseases
2National Center for Biotechnology Information
National Institutes of Health, Bethesda, MD 20892, USA
3The Catholic University of America, Washington, D.C. 20064, USA
4The Scripps Research Institute, La Jolla, CA 92037, USA
5Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA
Intimins and invasins are virulence factors produced
by pathogenic Gram-negative bacteria. They contain
C-terminal extracellular passenger domains that are
involved in adhesion to host cells and N-terminal
b domains that are embedded in the outer mem-
brane. Here, we identify the domain boundaries of
an E. coli intimin b domain and use this information
to solve its structure and the b domain structure
of a Y. pseudotuberculosis invasin. Both b domain
structures crystallized as monomers and reveal that
the previous range of residues assigned to the
b domain also includes a protease-resistant domain
that is part of the passenger. Additionally, we identify
146 nonredundant representative members of the
intimin/invasin family based on the boundaries of
the highly conserved intimin and invasin b domains.
We then use this set of sequences along with our
structural data to find and map the evolutionarily
constrained residues within the b domain.
The intimins and invasins (Int/Inv) constitute a family of outer
membrane proteins (OMPs) found in Gram-negative bacteria
that act as adhesins. Intimins are produced by ‘‘attaching
and effacing’’ (A/E) pathogens such as enterohemorrhagic
Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC).
A/E pathogens intimately adhere to epithelial cells that line the
intestinal wall and cause the formation of actin-rich lesions at
the site of interaction. These lesions are promoted by binding
of intimin to the translocated intimin receptor (Tir), a protein
that is produced by the bacteria, injected into the host cell,
and then integrated into the host cell membrane (Jerse et al.,
1990; Kenny et al., 1997). Invasins are homologous to intimins
and are produced by enteropathogenic species of the genus
Yersinia (Y. enterocolitica and Y. pseudotuberculosis) (Isberg
et al., 1987). Invasins bind to the b1-integrin superfamily of
proteins on the surface of eukaryotic host cells and trigger the
rearrangement of the host cell cytoskeleton that in turn leads
to the internalization of the bacteria (Isberg and Leong, 1990).
Int/Inv share a similar domain structure (Figures 1A–1D): (1) an
N-terminal signal peptide (SP) that targets them for secretion
through the Sec complex, (2) an internal transmembrane
‘‘b domain’’ that spans the outer membrane (OM), and (3) a
C-terminal passenger domain that mediates interactions with
host cells. Some Int/Inv also have an additional domain (LysM)
between their SP and b domain that is located in the periplasm
and predicted to bind peptidoglycan. The b domain is conserved
across family members, predicted to contain a b barrel, and is
necessary for the passenger domain to cross the OM (Touze ´
et al., 2004). Structures for portions of the passenger domain
have been solved by Batchelor et al. (2000), Hamburger et al.
(1999), and Luo et al. (2000). These structures form similar
elongated rods composed of repeated bacterial immunoglob-
ulin-like (BIG) domains and are capped at the C terminus by
C-type lectin-like domains.
Previous studies have indicated that autotransporters (ATs)
and Int/Inv share common characteristics that suggest they
are structurally related (Newman and Stathopoulos, 2004; Touze ´
et al., 2004): (1) both contain passenger domains that are
secreted into the extracellular space, and (2) both contain a b
domain that is essential for the translocation of the passenger
domain across the OM. However, there are some key differ-
ences: (1) Int/Inv have N-terminal b domains and C-terminal
passengers, whereas ATs have these domains inverted (the
passenger is N terminal, and the b domain is C terminal); and
(2) Int/Inv passenger domains are composed of BIG and
C-type lectin-like domains, whereas monomeric AT passenger
domains are predicted to fold as b helices in >97% of cases
(Junker et al., 2006).
For all solved monomeric AT b domain structures, a 12-
stranded b barrel is connected to a passenger domain by an
a-helical linker that spans the barrel pore (Barnard et al., 2012;
Oomen et al., 2004; Tajima et al., 2010; van den Berg, 2010;
Zhai et al., 2011). The Int/Inv b domains were also thought to
form b barrels in the OM, although the explicit domain bound-
aries and final number of b strands were unknown (Newman
and Stathopoulos, 2004; Touze ´ et al., 2004; Tsai et al., 2010).
Structure 20, 1233–1243, July 3, 2012 ª2012 Elsevier Ltd All rights reserved 1233
Here, we define the precise boundaries of intimin’s b domain
and identify a protease-resistant extracellular domain that is
not recognized as a BIG or C-type lectin-like domain. We
then use this information to solve the lipidic cubic phase
(LCP) X-ray crystal structures of the b domains of intimin from
EHEC strain O157:H7 and invasin from Y. pseudotuberculosis
strain IP 32953. We will refer to these subtypes simply as
intimin or invasin, respectively, from this point forward. Similar
to AT b domains, intimin and invasin are 12-stranded antipar-
allel b barrels containing linkers that span the barrel pore.
However, their structures also have significant differences
compared to the AT b domains. The linker that spans the barrel
pore adopts an extended conformation for intimin and invasin
rather than a-helical as seen for ATs. The extended linker
creates a large cavity on one side of the barrel pore. There is
also a periplasmic a helix between the barrel domain and linker
for intimin and invasin that is not present in ATs. To conclude,
we used the precise boundaries of the highly conserved
b domain to define the Int/Inv family and found groups of
evolutionarily constrained residues that cluster together in the
b domain structure.
RESULTS AND DISCUSSION
Mapping of the Intimin b Barrel Domain
Previously, residues 189–550 of EPEC intimin were observed to
form a protease-resistant domain that showed heat-modifiable
mobility when subjected to SDS-PAGE. b Barrel OMPs are
referred to as being heat modifiable if they remain folded in
SDS sample buffer at room temperature but then unfold upon
heating. The compact, folded barrel migrates faster during
SDS-PAGE than the unfolded/elongated protein. Additionally,
residues 189–550 of EPEC intimin were predicted to be rich
in b sheet structure (Touze ´ et al., 2004) and were thus proposed
to contain the transmembrane b barrel. To determine the
minimal domain of intimin that contains the b barrel, we made
N-terminal deletions in a construct expressing residues 189–
550 (HA-Int189–550) (Figure 2A) or C-terminal truncations of
a construct expressing residues 1–550 (HA-Int1–550) (Figure 2C).
We then tested to see if these truncation mutants showed
heat-modifiable mobility when subjected to SDS-PAGE. As ex-
lanes 1 and 2). The N-terminal deletion mutants HA-Int200–550
and HA-Int210–550also showed typical heat-modifiable mobility,
whereas HA-Int220–550did not (Figure 2B, lanes 3–8). Because
residues189–550 were previously foundto be protease resistant
(Touze ´ et al., 2004), these results suggest that residues 189–210
form a periplasmic, protease-resistant region upstream from
the intimin b barrel and that the N-terminal boundary of the
b barrel is located between residues 210 and 220. For the
C-terminal truncation mutants, we found that deletions up to
residue 450 (HA-Int1–450) did not affect intimin’s heat-modifiable
mobility (Figure 2D, lanes 1–8). However, the constructs (HA-
Int1–400, HA-Int1–411, and HA-Int1–430) with further C-terminal
truncations were no longer heat modifiable (Figure 2D, lanes
9–14). These results suggest that the C terminus of the b barrel
is between residues 430 and 450; however, this conclusion
was not correct, as will be discussed below.
Intimin requires the b barrel assembly machinery (BAM)
complex for proper insertion into the OM (Bodelo ´n et al.,
2009). The BAM complex recognizes unfolded b barrels in the
periplasm by interacting with a ‘‘signature sequence’’ located
in the final b strand of the barrel (Robert et al., 2006). This signa-
ture sequence is comprised of hydrophobic or aromatic amino
acids and includes the C-terminal residue of the final b strand.
A putative BAM signature sequence (L402YSMQFRYQF411)
was identified in the intimin sequence between residues 402
and 411. We hypothesized that this stretch of residues could
represent the last strand of the intimin b barrel. However, the
construct HA-Int1–411did not show heat-modifiable mobility as
would be expected for a fully folded b barrel (Figure 2D, lanes
11 and 12). One explanation for this result could be that, similar
to AT b domains, intimin has an a-helical linker that spans the
central pore of the b barrel and stabilizes it (Barnard et al.,
2007; Ieva et al., 2008). Indeed, PSIPRED calculations (Buchan
et al., 2010) predict that residues 412–449 of intimin have
Figure 1. Domains of Intimin and Invasin
The b domains of intimins (red barrel) and invasins (blue barrel) are predicted
to contain a b barrel and linker that would be embedded in the OM. The linker
likely passes through the barrel pore and connects the b barrel to the
passenger domain. The passenger domain is exposed to the extracellular
space and is comprised of repeating BIG domains and capped by a C-type
lectin-like domain. Regions of known structure are shown as ribbon diagrams,
and domain boundaries are indicated by amino position. Additionally, some
Int/Inv contain a predicted peptidoglycan-binding domain (LysM) between
their SP and b domain; this domain is shown only in (B) and is much more
prevalent among intimins.
(A and B) E. coli intimin. D0–D2 are BIG domains. The D3 domain is C-type
lectin-like domain. In (B) the domain boundaries defined by Touze ´ et al. (2004)
for the b domain (189–550) are shown above, and the actual domain bound-
aries for the b domain (210–449) and D00 domain (450–550) determined in
this study are shown below.
(C and D) Y. pseudotuberculosis invasin. The D1–D4 domains are BIG
domains. The D5 domain is C-type lectin-like domain. The boundaries of
invasin’s b domain were determined by aligning invasin with residues 189–550
of E. coli intimin.
b Domain Structures of Intimin and Invasin
1234 Structure 20, 1233–1243, July 3, 2012 ª2012 Elsevier Ltd All rights reserved
a-helical secondary structure. Thus, if a similar a-helical linker
region is located downstream of the last b strand of intimin’s
b barrel, this could explain why HA-Int1–411 and HA-Int1–430
were not observed to be heat modifiable. Using known AT
structures as guides, we predicted that intimin’s linker should
protrude from the pore of the b barrel at approximately residue
450 and that the region encompassing residues 450–550 should
be located in the extracellular space. To test this hypothesis,
we assessed the Proteinase K (PK) sensitivity of our C-terminal
truncation mutants in whole cells. It should be noted that the
region encompassing residues 450–550 of intimin is PK resistant
(Touze ´ et al., 2004). However, we reasoned that if the PK resis-
tance of this region is due to tight folding, then deleting portions
in contrast to HA-Int1–550, the constructs HA-Int1–530and HA-
Int1–500were sensitive to extracellularly added PK and were con-
verted to PK-resistant fragments, similar in size to HA-Int1–450
tected because they include the periplasmic domain, b barrel,
Figure 2. Residues 210–450 Define the Boundaries
of the Intimin b Domain
(A) The HA-Int construct is a full-length version of HA-
tagged intimin. The C terminus of this construct was
truncated at residue 550, and progressive N-terminal
deletions were then made between the HA tag and
(B) Heat-modifiable mobility assays for the N-terminal
deletion constructs are shown. Unfolded (UF) and folded
(F) intimin are indicated.
(C) C-terminal truncation constructs of intimin are illus-
(D) Heat-modifiable mobility assays for the C-terminal
truncation constructs are presented.
(E) Whole-cell PK-sensitivity assays of the C-terminal
truncation constructs are shown. Surface exposure of the
passenger is indicated by the appearance of a lower
molecular weight band compared to the full-length
construct when PK was added to the sample.
and a-helical linker of intimin and would there-
fore be shielded by the OM.
From these preliminary studies we concluded
that the intimin b domain likely contains a b
barrel comprised of residues 210–411 and an
a-helical linker that passes through the barrel
pore, connecting the barrel to the extracellular
passenger domain. The linker likely exits the
barrel pore at the cell surface near residue
450. The PK-resistant domain formed by resi-
dues 450–550, which we will refer to as domain
‘‘D00’’ from this point forward (Figure 1B), is
not recognized as a BIG or C-type lectin-like
domain by the Pfam database, suggesting that
its structure and function might be different
from the rest of the passenger domain.
Intimin b Domain Structure
Having determined that the intimin b barrel and
linker are located between residues 210 and
450, we cloned amino acids 208–449 from intimin into a pET9
vector. A PelB signal sequence was used to target intimin for
secretion, and for the purposes of purification, a 103His tag
and a TEV cleavage site were inserted between the PelB signal
sequence and the intimin-coding region (see Figure S1A
available online). This construct (103His-TEV-Int208–449) was
used to overexpress the intimin b domain, and we were able to
solubilize the recombinant protein from isolated membranes
with detergent. Purified intimin b domain showed heat-modifi-
able mobility when subjected to SDS-PAGE, indicating that it
is properly folded in the detergent micelle environment
(Figure S1C). We crystallized it from lipidic mesophases in the
C2221 space group and collected 1.85 A˚
(Table 1). Exhaustive attempts at structure solution via molecular
replacement using b barrel OMPs containing 10, 12, 14, or 16 b
strands proved unsuccessful. Thus, selenomethionine (SeMet)-
derivatized intimin was expressed, purified, and crystallized
using the 103His-TEV-Int208–449construct (Table 1). Although
analysis of the data with the program PHENIX (Adams et al.,
b Domain Structures of Intimin and Invasin
Structure 20, 1233–1243, July 3, 2012 ª2012 Elsevier Ltd All rights reserved 1235
2010) indicated that the anomalous signal was weak with a
resolution cutoff of 4.5 A˚, the program SHARP (Bricogne
et al., 2003) was able to identify five selenium sites, allowing
us to solve the structure. An initial model was manually built
into the experimentally determined electron density using the
program Coot (Emsley and Cowtan, 2004). This model was
then used to solve the high-resolution native intimin b domain
structure via molecular replacement using the program Phaser
(McCoy et al., 2007). Representative electron density for the
refined model is shown in Figure 3C. To our knowledge, intimin
is the first lipidic mesophase membrane protein crystal struc-
ture solved using 3-wavelength SeMet MAD data. Although
de novo phasing methods have been attempted for lipidic mes-
ophase crystals in the past using SeMet-derivatized proteins,
all attempts have proven unsuccessful thus far (Caffrey,
2011). This has been attributed to low-measurable anomalous
signal because mesophase crystals are generally small, weakly
diffracting, and often require averaging of several crystals to
obtain complete data sets. In our case a single mesophase
crystal was sufficient to collect three complete anomalous
data sets with a measurable anomalous signal due to SeMet
Previously, Touze ´ et al. (2004) showed that intimin could
dimerize and attributed this property to the b domain. Here,
our intimin construct crystallizes as a monomer (Figure 3A),
suggesting that either the dimeric form cannot be captured
under our purification and crystallization conditions or that our
construct does not possess the region or regions necessary
for dimerization. Indeed, dimerization was previously observed
for intimin residues 189–550 by Touze ´ et al. (2004), and here,
we only crystallized the b domain (b barrel plus linker, residues
208–449). Thus, it is possible that residues 189–207 and/or
residues in the D00 domain (residues 450–550) mediate dimer-
ization. There are also a few minor differences between our
construct and the protein used by Touze ´ et al., including an
uncleaved purification tag at the N terminus of our construct
and three naturally occurring amino acid differences between
The intimin b domain contains a 12-stranded b barrel and
central linker that passes through the barrel pore (Figures 3A
and 3B). Visible electron density begins at residue Gln 208 near
the N terminus of the b barrel and is continuous to the C-terminal
tified BAM signature sequence (L402YSMQFRYQF411) and is
immediately followed by a short a-helical region that would
face the periplasm. The protein chain then passes through the
central pore of the barrel in an extended conformation to form
a linker that would connect the barrel to the D00 domain of the
passenger. This is in contrast to the PSIPRED prediction of an
all contain a-helical linkers. The C-terminal residue, Lys 449, is
Extracellular loops 4 and 5 interact with this b strand to form
a small three-stranded b sheet above the barrel. As model
building progressed, electron density became evident for 13
monoolein molecules surrounding the b barrel, some fully
Table 1. Data Collection and Refinement Statistics for Native and SeMet-Derivatized Proteins
Data Collection Intimin NativeInvasin Native Intimin SeMet
a, b, c (A˚)
a, b, g (A˚)
117, 120, 39.179.4, 125, 65.1118, 120, 39.1 118, 120, 39.2118, 120, 39.2
90, 90, 9090, 90, 9090, 90, 90 90, 90, 9090, 90, 90
Rsym(%)8.8 (57.8) 13.3 (59.9)10.2 (24.7)11.4 (28.5)11.3 (28.9)
I/sI 21.1 (1.9) 10.5 (1.6) 10.8 (2.7)9.8 (2.7)9.3 (2.2)
Completeness (%)93.6 (71.5)92.7 (76.2)88.3 (49.2)93.0 (62.0)89.8 (51.3)
Redundancy5.1 (3.9)5.1 (2.9)3.5 (2.3) 3.5 (2.5)3.4 (2.1)
Unique reflections 22,224 14,111
Average B factor 32.037.1
No. of chains in ASU11
No. of protein atoms2,0031,961
No. of ligand atoms313283
Rmsd bond lengths0.0130.017
aValues in parentheses are for the highest resolution shell.
b Domain Structures of Intimin and Invasin
1236 Structure 20, 1233–1243, July 3, 2012 ª2012 Elsevier Ltd All rights reserved
in what appear to be individual ‘‘lipid channels’’ on the barrel’s
The barrel is ellipsoid in shape with a long axis of ?26 A˚and
a short axis of ?21 A˚when measured between Ca atoms. These
dimensions are similar to those of the AT EspP, and furthermore,
the EspP and intimin barrels superpose closely (Figures S3A
and S3B). When the linker is removed from the intimin barrel
pore, the equivalent pore diameter ranges from ?6 to ?10 A˚
when measured by the program HOLE using the Connolly option
(Figures S3A and S3C) (Smart et al., 1996). Similarly, the equiva-
lent pore diameter of precleavage EspP with its linker removed
ranges from ?5 to ?11 A˚. Because the side chains within the
barrel pore can sample different conformations in vivo but are
fixed during analysis, the equivalent pore diameter measured
by HOLE likely underestimates the maximum size the pore can
attain. Nevertheless, a pore diameter of ?10 A˚would preclude
the passage of protein domains with tertiary structure. Because
ATs and the Int/Inv family likely share a common translocation
mechanism, and elements that would not fit inside an ?10 A˚
pore have been seen to be efficiently secreted for ATs (Jong
et al., 2007; Leyton et al., 2011; Saurı ´ et al., 2012; Skillman
et al., 2005), the barrel pore seen in the intimin structure and
the AT structures probably does not accurately depict the active
translocation channel. It should be noted that several proteins
containing disulfide bonds have been fused to intimin (Adams
et al., 2005; Wentzel et al., 2001) and tested for passenger trans-
location to the cell surface. Similar to the AT studies, some of
these proteins were translocated efficiently, whereas others
were not. However, none of the fusions that were transported
efficiently by intimin were tested to see if their cysteines were
more accessible on the cell surface in a dsbA?strain compared
to a wild-type strain. Increased accessibility of surface-exposed
cysteines in a dsbA?strain would suggest that the disulfide
bonds were formed in the periplasm and that the passenger
fusions were at least partially folded during translocation. For
a recent review discussing AT translocation models, please refer
to Leyton et al. (2012).
The linker primarily contacts one side of the barrel pore, form-
ing a large network of hydrogen bonds and salt bridge interac-
tions with residues from the barrel wall (Figure 4A). Due to this
asymmetry a large cavity (1,901.9 A˚3) (Dundas et al., 2006) is
created on the opposite side of the barrel pore (Figures 4B and
4C). This cavity is almost completely enclosed by extracellular
loop 5 and the periplasmic a helix. However, small channels
(?3 A˚diameter) that connect to the large cavity create pathways
that traverse the length of the barrel when visualized with the
program CAVER (Bene? s et al., 2010). To see if the large cavity
and these small channels could form a conductance pathway,
we reconstituted purified intimin (103His-TEV-Int208–449) into
a lipid bilayer and applied various voltages. However, no
conductance increases were observed (Figure S3D), suggesting
that intimin cannot form an open channel. These results agree
with those of Touze ´ et al. (2004). When they similarly tested an
intimin construct containing residues 1–550, they observed no
changes in conductance as well. It is possible that this empty
space could serve as a translocation pathway for the passenger
(assuming loop 5 is flexible and the periplasmic a helix is in
a different conformation during translocation). However, this
can expand) because there is evidence, as mentioned above,
that ATs and Int/Inv can transport passengers containing
elements that would not fit inside a 10 A˚pore (Adams et al.,
2005; Jong et al., 2007; Leyton et al., 2011; Saurı ´ et al., 2012;
Skillman et al., 2005; Wentzel et al., 2001).
Mutational Analysis of Intimin’s Linker and Periplasmic
For the AT EspP, previous studies have shown that the portionof
its a-helical linker near the periplasmic side of the barrel pore is
Figure 3. Structure of the Intimin b Domain
(A) Stereo ribbon diagram of the intimin construct 103His-TEV-Int208–449is
illustrated. Strands, salmon; loops, yellow-green; helices, light orange. The
extended portion of the linker inside the barrel pore is highlighted in cyan.
(B) Two-dimensional representation of the b domain imposed onto its
secondary structure is shown. Loop residues, green circles; extended linker
residues, cyan circles; a helix residues, orange hexagons; b strand residues,
red (central pore-facing) or blue (outward-facing) arrows.
(C) Stereo diagram of b strands 5–8 of intimin in stick form with the 2Fo-Fc
electron density (blue mesh) shown at a contour level of 1s.
See also Figure S2.
b Domain Structures of Intimin and Invasin
Structure 20, 1233–1243, July 3, 2012 ª2012 Elsevier Ltd All rights reserved 1237
essential for proper folding of the barrel and passenger translo-
cation (Barnard et al., 2007; Ieva et al., 2008). Intimin’s extended
linker is also important for stability of its barrel, as indicated by
the lack of heat modifiability observed for the HA-Int1–430
construct (Figure 2D, lanes9and 10). Thus, we decided to deter-
mine the minimum length of intimin’s linker necessary for forma-
tion of a heat-modifiable b domain. Using the HA-Int1–450
construct, we constructed deletion mutants where the C
terminus was truncated two residues at a time. The heat-modifi-
able mobility of each of these mutants was then assayed, and
typical shifts in migration were seen until residues Leu 437 and
Val 438 were deleted (Figure 5A, lanes 1–14). At this point,
further truncations in the linker led to the production of
nonheat-modifiable products (Figure 5A, lanes 15–18). Both
Leu 437 and Val 438 form hydrophobic interactions with the
barrel wall, and the solvation energy changes for these residues
upon formation oftheinterface betweenthelinkerandbarrelwall
are ?1.98 and ?1.56 kcal/mol, respectively, as estimated using
the PISA web server (Krissinel and Henrick, 2007).
We also investigated the contribution to b domain stability of
three salt bridges formed between the linker and the barrel
wall. Linker residues Arg 434, Asp 436, and Arg 440 form
charge-charge interactions with barrel wall residues Asp 236,
Lys 301, and Asp 279, respectively (Figure S4A), and are highly
conserved in the Int/Inv family (Figure S6). We disrupted these
interactions with alanine substitutions in the HA-Int1–530
construct and then tested the mutants for heat modifiability of
their b domains and PK accessibility of their passenger domains
(Figures S4B and S4C). We found that disrupting any one of
these interactions had no effect compared to the HA-Int1–530
wild-type construct, suggesting that multiple interactions
between the barrel and linker need to be perturbed to signifi-
cantly alter the stability of the b domain or translocation of the
A novel structural feature found in intimin, but not ATs thus far,
is the periplasmic a helix that lines the bottom side of the barrel.
To test whether this region is important for forming a stabile
b barrel or passenger translocation, we replaced this domain
(residues 414–433) with a short linker consisting of two glycine
residues to create the HA-Int1–530D414–433 construct. Unex-
pectedly, this construct showed wild-type heat modifiability
and PK sensitivity, demonstrating that its b barrel was properly
folded and that the truncated passenger domain was secreted
(Figures 5B and 5C).
Invasin b Domain Structure
To solve the b domain of another Int/Inv family member, we used
the intimin structure as a guide to solve Y. pseudotuberculosis
invasin. First, we aligned the residues included in the structure
aligned with ?50% identity. Similar to intimin, these residues
were then cloned behind a PelB signal sequence followed by
a 103His tag and TEV protease cleavage site to create the
construct, 103His-TEV-Inv147–390(Figure S1B). However, initial
expression trials using this construct led to the production of
invasin inclusion bodies and protein that was associated with
the OM, but not extractable with detergents. One explanation
for this result could be that invasin does not contain a typical
E. coli BAM signature sequence. For invasin this sequence
(Q343WNLQMNYRL352) lacks the conserved Phe/Trp residue
normally found at the last position. We hypothesized that
changing this sequence for invasin so that it more closely
matched the E. coli BAM signature sequence would result in
insertion into the OM. Indeed, after mutating Leu 352 to a Phe
residue, we were able to purify milligram quantities of properly
folded and inserted invasin (Figure S1D). The invasin b domain
was crystallized in monoolein lipidic mesophases, and its struc-
ture was solved to 2.3 A˚resolution by molecular replacement,
using the high-resolution LCP structure of the intimin b domain
(Table 1). As expected, the invasin b domain is monomeric and
composed of a 12-stranded b barrel and linker (Figure 6A). The
b domains of invasin and intimin are structurally very similar
with an rmsd for their Ca carbons of 0.70 A˚(Figure 6B), whereas
the barrel domains (linker and portions of some extracellular
loops removed) of invasin and EspP are less similar with
tural alignment. One difference between the intimin and invasin
structures lies in the first extracellular loop. For invasin this loop
is in a ‘‘flipped-out’’ conformation compared to intimin due to
a two amino acid insertion in this region (Figures 6B and S6).
Figure 4. Intimin’s Linker Contacts One Side of the Barrel Wall
(A) The intimin b barrel is shown in stereo ribbon format. Strands, salmon;
loops, yellow-green. Hydrogen bonds between residues of the barrel wall
(salmon sticks) and residues of the linker (cyan sticks) are shown as dashed
(B) Cavity within the b barrel of intimin is shown as a black surface.
(C) Same as (B) but rotated by 270?about the x axis.
See also Figure S3.
b Domain Structures of Intimin and Invasin
1238 Structure 20, 1233–1243, July 3, 2012 ª2012 Elsevier Ltd All rights reserved
Bioinformatic Analysis of the Intimin and Invasin
b Domain Structures
The b barrel and linker regions of intimin and invasin share 50%
sequence identity and 65% sequence similarity when their
sequences are aligned via the NCBI BLAST server. On the other
hand, their extracellular passenger domains share only 25%–
30% sequence identity with 37%–50% sequence similarity.
Additionally, a recent study by Tsai et al. (2010) on homologs
of intimin and invasin confirmed that the b domain is the most
conserved part of the protein. Because we identified the exact
boundaries for the b barrel and linker portions of intimin and
invasin, we tried searching for new Int/Inv family members using
only residues 208–449 of intimin and 147–390 of invasin rather
than their entire protein sequences. BLAST queries against the
nonredundant database using these regions identified 769
protein sequences with an E value cutoff of 10?2. After removing
sequence redundancy and additional filtering (see Supplemental
Experimental Procedures), we identified 146 representative
members of the Int/Inv family, significantly more than the 69
146 sequences by the NCBI Taxonomy Browser indicated that
they belonged to 83 Gram-negative bacterial species and 1
uncultured bacterium. Of the 83 Gram-negative bacterial
species identified, 76 were g-proteobacterial, 5 were b-proteo-
bacterial, 1 was a-proteobacterial, and 1 was from Chlamydia
(Table S2; Figure S5). Multiple sequence alignment (MSA) of
these 146 proteins was then performed using the programs
COBALT (Papadopoulos and Agarwala, 2007) and MUSCLE
(Edgar, 2004) (Figure S6). A large number of highly conserved
residues were observed in the MSA of the b domain. Residues
showing 100% identity across all 146 representative members
are located in an interior section of the barrel wall that is close
to the linker and in the linker itself (Figures S6 and S7). Moreover,
Figure 5. Deletion Analysis of Intimin’s
Linker and Periplasmic a Helix
(A) Heat-modifiable mobility assays for the linker
deletion constructs are shown. C-terminal trun-
cations were made starting with residue 450 of the
HA-Int1–450construct. Each truncation removed
two residues. Unfolded (UF) and folded (F) forms
of the b domain are indicated.
(B) Heat-modifiable mobility
constructs HA-Int1–530and HA-Int1–530D414–433
are presented. In HA-Int1–530D414–433, the peri-
plasmic a helix was replaced with two glycines.
This construct retains heat-modifiable mobility.
(C) Whole-cell PK-sensitivity assays for the
constructs in (B). HA-Int1–530D414–433 trans-
locates its passenger to the cell surface when the
periplasmic a helix is replaced by two glycines.
See also Figure S4.
the MSA shows that b strands are struc-
turally conserved in the Int/Inv family,
and sporadic insertions and deletions
are almost exclusively located in the
loop regions of the b domain (Figure S6).
In addition to identifying conserved
residues in the Int/Inv family, we also
searched forresiduesthat coevolve.Residuesundergo coevolu-
tion when mutation in one residue triggers a mutation in another
residue. Thesesimultaneous changesallow a proteinto maintain
Figure 6. Structure of the Invasin b Domain
(A) Ribbon diagram of the invasin b domain structure with light-blue strands,
yellow helices, and violet loops is illustrated. The extended portion of the linker
inside the barrel pore is highlighted green.
(B) Superposition of the invasin and intimin b domains is shown. Invasin is
colored as in (A), and intimin is colored as in Figure 3A. Extracellular loop 1 for
intimin and invasin is marked with an asterisk.
See also Figure S1.
b Domain Structures of Intimin and Invasin
Structure 20, 1233–1243, July 3, 2012 ª2012 Elsevier Ltd All rights reserved 1239
its conformational and functional stability during evolution. For
example a mutation that has a negative impact on the function
of the protein will be accommodated by a compensatory muta-
tion at a nearby site or sites such that the function of the protein
is preserved. Complementing conservation analysis, coevolu-
tion analysis allows for detecting functionally and structurally
important sites that are not necessarily conserved. In particular
although such an analysis is based on sequence only, it often
reveals relations between residues that are close in space rather
than sequence. Coevolving pairs or groups of residues can be
identified by searching for pairs of residues whose mutations
are correlated more highly than expected from common evolu-
tionary history of the whole proteins/domains. After coevolution
analysis with a slightly modified version of the Mlp method (Dunn
et al., 2008), we found that the b domain of the Int/Inv family
contains numerous pairs of residues with significant coevolution
(Table S3). A large group of coevolving residues forms a patch of
spatially close residues that cluster on one side of the b domain
(Figures 7A and 7C). The majority of these residues point toward
the barrel pore from the barrel wall or are in extracellular loops
near the barrel pore, whereas two residues (Ile 444 and Lys
449) are in the linker. Additionally, this coevolving patch is
located near the cluster of residues that are 100% identical
Similar to the coevolving patch, these identical residues all point
toward the barrel pore, whereas one residue (Arg 440) is part of
Figure 7. Coevolving Residues for the Int/Inv Family
(A) A patch ofcoevolving residuesfor the Int/Invfamily mapped ontointimin and shown assticks is presented.Acidic residues are colored salmon, basic residues
are colored blue, and all others are colored yellow. Portions of the intimin structure have been removed to more clearly show the patch.
(B) Selected coevolving pairs of residues for the Int/Inv family mapped onto intimin and shown as sticks are illustrated. Only coevolving pairs near to each other in
the structure are shown. The pairs are color coordinated and have their side chains shown as sticks.
(C) Circle diagram for the coevolving patch showing connectivity of the network is presented. Colors are coordinated with (A). Only connections between
coevolving residues less than 10 A˚apart are shown.
(D) Circle diagram for coevolving pairs is shown. Colors for pairs are coordinated with (B).
See also Figure S7 and Table S3.
b Domain Structures of Intimin and Invasin
1240 Structure 20, 1233–1243, July 3, 2012 ª2012 Elsevier Ltd All rights reserved
the linker. Because the residues in the coevolving patch and the
identical residues are all located on the side of the b domain that
is near the linker or in the linker itself, this suggests that
these regions of the b domain are critical for Int/Inv biogenesis.
However, we made deletions and point mutations (R440A,
D279A) in this region and saw no effect on passenger transloca-
tion or heat-modifiable mobility (Figures 5A and S4). Together,
these data suggest that several residues, whether they are
part of the coevolving patch or are highly conserved, need
to be mutated to obtain a significant stability or translocation
Coevolving pairs of residues that were not part of the large co-
evolving group (Table S3) were also found throughout the
b domain (Figures 7B and 7D). The majority of the pairs of co-
evolving residues are close to one another in the structure,
whereas some are also separated by large distances in the
sequence. For example, Glu 324 and Arg 254 are separated by
70amino acids in sequence, yet theywere predicted to coevolve
in our analysis and were then found to form a salt bridge in the
intimin structure (Figures 7A and 7C).
Here, we present the first b domain structures of two archetypal
members of the Int/Inv family of adhesins. Previous work identi-
fied the b domain of intimin as including residues 189–550;
however, we show that residues 450–500 actually form
a protease-resistant domain (D00) that is part of the extracellular
lectin-like domain, and its function is unknown. This domain
could be involved in dimerization because a proteolytic fragment
of intimin containing residues 189–550 has been shown to
dimerize, whereas our shorter construct containing residues
208–449 cannot. Another possible function for the D00 domain
that could be explored in future studies is its role in passenger
translocation. For ATs and intimin this region of the passenger
is translocated to the cell surface first, whereas the majority of
the passenger is still in the periplasm. Furthermore, for ATs it
has been shown that proper folding of this region is then neces-
sary fortranslocation of the full-length passenger to the extracel-
and invasin b domains also include novel features not seen in
ATs, including an extended linker, a large cavity inside the
barrel, and a periplasmic a helix. Similar to ATs, the portion of
the linker closest to the periplasm is important for stability of
the b barrel. However, the periplasmic a helix can be replaced
by two glycines with little effect. One possible function for this
a helix could be to seal the large cavity inside the barrel from
the periplasmic side. Finally, we used the precise boundaries
of the highly conserved intimin and invasin b domains to
identify 146 nonredundant representative members of the Int/
Inv family. We then used this set of sequences along with our
structural data to find and map the evolutionarily constrained
residues within the b domain. Evolutionary analysis of these
sequences pointed to many conserved residues and coevolving
groups of residues. In particular, perfect conservation of many
residues in the interior of the barrel wall closest to the linker
together with coevolution of the linker and wall residues in the
same portion of the barrel suggests the functional and structural
importance of this region.
b Domain Stability Assay
Details on plasmid construction are available in the Supplemental Experi-
mental Procedures. To assay protein heat-modifiable mobility, E. coli AD202
cells transformed with the appropriate plasmids were grown in M9 minimal
medium containing all the amino acids except Metand Cys and supplemented
with 100 mg/ml ampicillin or 50 mg/ml kanamycin. At an OD600nm of 0.2,
synthesis of the protein was induced by adding 100 mM IPTG. After 30 min
of induction, cells were harvested by centrifugation (3,000 rpm, 10 min, 4?C),
resuspended in 13 PBS containing 1 mM PMSF (phenylmethanesulfonylfluor-
ide) to an OD600nmof 10, and sonicated until clear. N-octyl b-D-glucopyrano-
side was then added to the lysate to a final concentration of 1% (w/v), and
samples were incubated for 15 min at room temperature. Unbroken cells
and insoluble materials were then removed by centrifugation (5,000 3 g,
10 min, 4?C). The supernatant was collected, and 15 ml was mixed with an
equal volume of 23 SDS-PAGEloading buffer. These samples were incubated
at the indicated temperatures for 10 min before separation by SDS-PAGE.
Results were visualized via western blot with an anti-HA antibody.
Passenger Secretion Assay
To assay passenger domain accessibility to PK in whole cells, E. coli AD202
cells transformed with the appropriate plasmids were grown and induced as
described for the b domain stability assay. After 30 min of induction, two
1 ml aliquots were taken, and the cells were harvested by centrifugation
(3,000 rpm, 10 min, 4?C) and then resuspended in 1 ml of PBS. One sample
was left untreated (?PK), whereas the other was treated (+PK) with 10 ml
of 20 mg/ml PK (Calbiochem). After 30 min of incubation at 4?C, 10 ml of
100 mM PMSF was added, and the samples were TCA (trichloroacetic acid)
precipitated and centrifuged (13,000 rpm, 4?C, 10 min). The pellets were
resuspended in Trix buffer (15% glycerol, 200 mM TRIS, 15 mM EDTA,
4% SDS, 10 mM DTT), mixed with SDS-PAGE loading buffer, and resolved
by SDS-PAGE. Results were visualized via western blot using an anti-HA
Lipidic Mesophase Crystallization
Detailed expression and purification protocols are available in the Supple-
mental Experimental Procedures. Purified native or SeMet-labeled 103His-
TEV-Int208–449and 103His-TEV-Inv147–390in the size exclusion chromatog-
raphy buffer were concentrated to 40 and 28 mg/ml, respectively, and then
diluted with dH2O to 20 mg/ml. Monoolein (Nu-Chek Prep) was melted at
42?C and then 60 ml of molten monoolein was mixed with 40 ml of protein at
20 mg/ml in a coupled syringe apparatus as described previously by Caffrey
and Cherezov (2009). The final concentration of protein in the lipidic meso-
phase was 8 mg/ml. Lipidic mesophase crystallization trials were set up
using 96-well Laminex bases (Molecular Dimensions) and a Mosquito LCP
robot (TTP Labtech) and then sealed with Laminex film covers (Molecular
Dimensions). Each well contained 100 nl of lipidic mesophase and 750 nl of
well solution. The plates were incubated at 21?C, and crystals were visible
within 1–2 days. Final optimized well solution for native 103His-TEV-Int208–449
contained 0.1 M sodium citrate (pH 4.5–5.5), 0.05–0.1 M NaCl, 0.1–0.15 M
MgCl2, and 30%–34% PEG-400. SeMet-103His-TEV-Int208–449 optimized
well solution contained 0.1 M phosphate-citrate (pH 4.2), 30%–40% ethanol,
and 1%–3% PEG-1000. 103His-TEV-Inv147–390optimized well solution con-
tained 0.05 M sodium citrate (pH 3.8–4.4), 0.2 M Li2SO4, and 23%–35%
Data Collection, Structure Determination, and Refinement
Crystals were directly harvested from LCP using LithoLoops from Molecular
Dimensions and flash frozen in liquid nitrogen until data collection. All data
were collected at the GM/CA-CAT 23ID-B and 23ID-D beamlines at the
Advanced Photon Source within the Argonne National Laboratory. All data
were processed using HKL2000 (Otwinowski and Minor, 1997). MAD phasing
with the SeMet data sets (Peak, Inflection, and H.E. Remote) was performed
using SHARP (Bricogne et al., 2003), which found five selenium sites and
resulted ininterpretable electron densitymaps afterdensity modification using
SOLOMON (Abrahams and Leslie, 1996). An initial model was built into the
electron density maps and then used to solve the high-resolution native
b Domain Structures of Intimin and Invasin
Structure 20, 1233–1243, July 3, 2012 ª2012 Elsevier Ltd All rights reserved 1241
103His-TEV-Int208–449data set via molecular replacement using the program
Phaser (McCoy et al., 2007). The 103His-TEV-Inv147–390structure was solved
via molecular replacement using Phaser with the high-resolution native
103His-TEV-Int208–449structure as the search model. The structures were
refined using the programs PHENIX (Adams et al., 2010) and REFMAC
(Murshudov et al., 1997) interspersed with rounds of model building using
Coot (Emsley and Cowtan, 2004). Data collection and refinement statistics
are available in Table 1. Each data set was obtained from a single crystal. All
figures containing molecular graphics were prepared using the program
PyMOL (Schro ¨dinger).
Bioinformatic Analysis of the Intimin and Invasin b Domains
BLAST (Altschul et al., 1990) searches using E value cutoffs of 0.01 were per-
formed against the nonredundant protein database with the intimin (GenInfo
Identifier, 118201527) and invasin (GenInfo Identifier, 51596004) b barrel
sequences. The presence of a b barrel in each protein from the search was
confirmed using the Conserved Domain Database (Marchler-Bauer et al.,
2011). Redundant sequences with >95% sequence identity were removed
using the program CD-HIT (Li and Godzik, 2006). The remaining sequences
were compiled into a MSA using the programs COBALT (Papadopoulos and
Agarwala, 2007) and MUSCLE (Edgar, 2004), and the MSA was further
improved by manual inspection. The final MSA contained 146 nonredundant
representative members of the Int/Inv family. Coevolving residues were iden-
tified using the modified Mlp method (Dunn et al., 2008). Detailed methods are
available in the Supplemental Experimental Procedures.
The coordinates and structure factors have been deposited in the Protein Data
Bank as entries 4E1S for intimin and 4E1T for invasin.
Supplemental Information includes seven figures, three tables, and Supple-
mental Experimental Procedures and can be found with this article online at
We thank the members of the user support staff at the GM/CA-CAT beamline
at the Advanced Photon Source, which is supported by National Cancer
Grant Y1-GM-1104, for their assistance during data collection. This work was
supported by the Intramural Research Program of the National Institute of
Diabetes & Digestive & Kidney Diseases (to J.W.F., N.N., T.J.B., and S.K.B)
and the National Library of Medicine (to D.W. and T.M.P.) of the National Insti-
GM073197 (to W.L. and V.C.); by NIH training Grant GM008572 (to E.U.); and
by Polish National Science Center Grant 2011/01/B/ST6/02777 (to D.W.).
Received: March 3, 2012
Revised: April 21, 2012
Accepted: April 25, 2012
Published online: May 31, 2012
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b Domain Structures of Intimin and Invasin
Structure 20, 1233–1243, July 3, 2012 ª2012 Elsevier Ltd All rights reserved 1243