Structural insights into Helicobacter pylori oncoprotein
CagA interaction with β1 integrin
Burcu Kaplan-Türköza,1, Luisa F. Jiménez-Sotob,1, Cyril Dianc, Claudia Ertlb, Han Remautd,e, Arthur Louchea,
Tommaso Tosic, Rainer Haasb,2, and Laurent Terradota,2
a
Centre National de la Recherche Scientifique-Ligue Contre le Cancer, ATIP Avenir Group, Institut de Biologie et Chimie des Protéines, Unité Mixte de
Recherche 5086, Université Lyon, Lyon F-69367, France; bMax von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Department of
Bacteriology, Ludwig-Maximilians-Universität, D-80336 Munich, Germany; cInstitut de Biologie Structurale J. P. Ebel, F-38027 Grenoble Cedex, France;
d
Structural Biology Brussels, Vrije Universiteit Brussels, 1050 Brussels, Belgium; and eStructural and Molecular Microbiology, Vlaams Instituut voor
Biotechnologie (VIB) Department of Structural Biology, VIB, 1050 Brussels, Belgium
Edited* by Scott J. Hultgren, Washington University School of Medicine, St. Louis, MO, and approved July 31, 2012 (received for review April 16, 2012)
Infection with the gastric pathogen Helicobacter pylori is a risk
factor for the development of gastric cancer. Pathogenic strains
of H. pylori carry a type IV secretion system (T4SS) responsible
for the injection of the oncoprotein CagA into host cells. H. pylori
and its cag-T4SS exploit α5β1 integrin as a receptor for CagA trans-
location. Injected CagA localizes to the inner leaflet of the host cell
membrane, where it hijacks host cell signaling and induces cyto-
skeleton reorganization. Here we describe the crystal structure of
the N-terminal ∼100-kDa subdomain of CagA at 3.6 Å that unveils
a unique combination of folds. The core domain of the protein
consists of an extended single-layer β-sheet stabilized by two in-
dependent helical subdomains. The core is followed by a long helix
that forms a four-helix helical bundle with the C-terminal domain.
Mapping of conserved regions in a set of CagA sequences identi-
fied four conserved surface-exposed patches (CSP1–4), which rep-
resent putative hot-spots for protein–protein interactions. The
proximal part of the single-layer β-sheet, covering CSP4, is in-
volved in specific binding of CagA to the β1 integrin, as deter-
mined by yeast two-hybrid and in vivo competition assays in H.
pylori cell-culture infection studies. These data provide a structural
basis for the first step of CagA internalization into host cells and
suggest that CagA uses a previously undescribed mechanism to
bind β1 integrin to mediate its own translocation.
virulence factor | X-ray crystallography | oncogene | pathogenicity |
gastric ulcer
F ifty percent of the world population is infected by Helicobacter
pylori, which causes severe gastric pathologies, including pep-
tic ulceration and gastric cancer (1). The most virulent strains
harbor the cag pathogenicity island (cag-PAI), a 40-kb DNA
fragment that encodes the cytotoxin CagA and a type IV secretion
system (cag-T4SS). CagA is a unique protein not found in any
other bacteria besides H. pylori. Transgenic mice expressing the
cytotoxin develop gastric polyps and adenocarcinomas, estab-
lishing CagA as a unique bacterial oncoprotein (2). Upon contact
with the gastric epithelial cells, H. pylori delivers CagA via the cag-
T4SS, a needle-like molecular appendage spanning both bacterial
membranes prolonged by a large extracellular pilus (3, 4). Several
proteins, such as CagL, CagY, and CagA, decorate the H. pylori
cag-T4SS pilus and use α5β1 integrin at focal adhesions as a re-
ceptor to target CagA delivery (5–7). However, the mechanism of
CagA translocation across the eukaryotic membrane via the cag-
T4SS conduit has not been established.
Once injected, CagA localizes at the inner leaflet of the plasma
membrane at cell-adhesion junctions, where it activates a multi-
faceted attack on host cell signaling. At adherens junctions, CagA
forms a complex with cytoskeleton proteins, in particular with E-
cadherin (8, 9). Based on high-throughput screening for soluble
fragments (10) and on in vivo proteolysis data, CagA can be de-
scribed as a protein consisting of two functional domains (11).
Much attention has been paid to the role of the C-terminal do-
main (residues 885–1,186). Indeed, this segment bears three Glu-
14640–14645 | PNAS | September 4, 2012 | vol. 109 | no. 36
Pro-Ile-Tyr-Ala (EPIYA) motifs that become tyrosine phos-
phorylated by Src and Abl kinases in eukaryotic cells (Fig. 1A).
Strains of Western origin harbor three different EPIYA phos-
phorylation motifs, namely A, B, and C (sometimes multiple C
motifs) and East Asian strains display A, B, and D motifs (12).
Phosphorylated CagA then binds and activates Src homology
2 domain phosphatase (SHP2) via its SH2 domains, leading to
dephosphorylation and inactivation of Src family kinases, result-
ing in morphological transformation and dramatic cytoskeletal
rearrangements in later stages of infection (13). Inactivation of
Src family kinases results in dephosphorylation of vinculin, dis-
rupting vinculin interaction with Arp2/3, which leads to de-
creased lamellipodia formation and decreased number of focal
adhesions. Other CagA effects are phosphorylation-independent.
In particular, specific sequences (named MKI) (Fig. 1A) located
in the CagA C terminus inhibit Par1b/MARK2 kinase activity
via mimicry of the enzyme’s natural substrate (14). The in-
hibition of the PAR1b/MARK2 perturbs atypical PKC signaling,
which results in disruption of tight junctions and loss of cell
polarity (15).
Recent studies have focused on the N-terminal portion of CagA
(residues 1–884, CagA1–884) (Fig. 1A). This domain interacts with
intracellular partners, such as ASPP2 (16), RUNX3 (17), TAK1,
and TRAF6 (18), and thus plays an important role in the de-
velopment of gastric cancer. Moreover, CagA1–884 contains resi-
dues that bind to the ectodomain of α5β1 integrin, but the
relevance of this binding for the process of CagA translocation, if
any, was not known (6). Here we present the crystal structure of
CagA1–884, which reveals a unique combination of domains. The
analysis of the structural similarity indicates that the core domain
resembles the outer surface protein A from Borrelia burgdorferi
but the C-terminal helical domain shows some resemblance to
eukaryotic cytoskeleton proteins. A specific single-layer β-sheet
region (SLB) was identified to act as the functional binding do-
main of CagA to β1 integrin. We further show that this specific
interaction with β1 integrin is essential for the process of CagA
translocation.
Author contributions: B.K.-T., L.F.J.-S., R.H., and L.T. designed research; B.K.-T., L.F.J.-S., C.D.,
C.E., H.R., A.L., and L.T. performed research; T.T. contributed new reagents/analytic tools;
B.K.-T., L.F.J.-S., C.E., H.R., and R.H. analyzed data; and B.K.-T., L.F.J.-S., H.R., R.H., and L.T.
wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org (PDB ID code 4GOH).
1
B.K.-T. and L.F.J.-S. contributed equally to this work.
2
To whom correspondence may be addressed. E-mail: haas@mvp.uni-muenchen.de or
laurent.terradot@ibcp.fr.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1206098109/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1206098109

Results
Overall Structure of CagA1–884. To gain structural insight into the
mode of action of CagA, we solved the crystal structure of CagA1–884
to 3.6Å (Rfactor/Rfree of 0.32/0.34). The protein crystallized in the
space group P41212 with one molecule per asymmetric unit.
CagA1–884 has an elongated overall structure consisting of four
domains arranged in a “crescent moon” shape (Fig. 1 B and C, and
Fig. S1). Even though seven α-helices are clearly visible (Fig. 1B),
the N-terminal domain D1 was not included in the refined model
because poor quality of the electron density map in this area of the
protein prevented unambiguous model building and assignment
of the sequence. A linker region follows D1, which we estimate to
be between residues 270 and 300, based on domain topology and
limited proteolysis (SI Materials and Methods and Fig. S2). The
second domain, D2, forms the central core of CagA1–884 structure,
Kaplan-Türköz et al.
Fig. 1. Overall structure of CagA N-terminal do-
main. (A) Schematic representation of CagA (Western
strain 26695). Boxes indicate the domain definition
derived from the CagA1–884 X-ray structure, with D1
colored in red, the SLB in pink, subdomain D2′ in
cyan, D2′′ in blue, D3 in green, and D4 in yellow.
The positions of the EPIYA motifs are indicated by
A, B, and C and those of PAR1-MARK kinase binding
motifs (14) by MKI1 and MKI2. Bars indicate the
positions of the protein fragments used in the study.
(B) Ribbon representation of the structure of
CagA1–884 colored according to A. (C) Topology di-
agram of CagA1–884 structure.
spanning residues 305–642 and containing three subdomains. The
major part of D2 consists of 11 antiparallel strands (β1–β5 and β8–
β13) forming a SLB that has a left-hand twist and is curved at
strand β8 (Fig. 1B). Interestingly, this SLB is structurally similar to
that of the outer surface protein OspA from B. burgdorferi (19)
(DALI Z-score: 5.3) (Fig. S3). The homology region is limited to
strands β2–β5 and β8–β11 of CagA that superimpose well to
strands β6–β13 of OspA (Fig. S3). The resulting structure-based
sequence comparison indicates that the first four strands are the
most similar. As a consequence, the surface properties of the
proximal parts of the two SLBs have comparable hydrophobic
properties (Fig. S3).
A compact subdomain (D2′), formed by a β-hairpin (β6, β7)
and helices α8 to α10, is inserted between strand β5 and β8 and
buttresses the lower half of the β-sheet (Fig. 1 B and C, and Fig.
Fig. 2. Structure-based sequence alignment of
CagA1–884. Sequences of CagA from strain 26695
(46), V225 (47), HP-No20, and HP-No31 (48), were
selected from the multiple alignment of 80 sequen-
ces generated by CONSURF (49) to illustrate CagA
diversity. Sequence numbering corresponds to CagA
from strain 26695. Conserved and invariant residues
(among 80 CagA sequences) exposed on the surface
of CagA1–884 crystal structure are shaded in magenta
and red, respectively. The exact sequence of strand
β1 could not be determined; the mostly likely can-
MICROBIOLOGY
didate sequence is indicated in black. Secondary
structure elements are indicated above the sequen-
ces and colored according to the domain: D2′, cyan;
SLB, pink; D2′′, purple; D3, green; and D4, yellow.
Blue dots are positioned below residues R624 and
R626, involved in PS binding (25).
PNAS | September 4, 2012 | vol. 109 | no. 36 | 14641
S1). Finally, the SLB is followed by a helical domain (D2′′)
consisting of the short helix α11 and two helices (α12 and α13)
that adopt a hairpin structure that packs against the convex face
of the SLB and stabilizes the upper half of the β-sheet (Figs. 1B
and 2). D3 is a 90 Å-long kinked amphipathic helix (α14) that
protrudes away from the core. The N-terminal moiety of α14
engages into a hydrophobic pocket formed by helices α11, α12,
and α13 of domain D2′′ (Figs. 1B and 2, and Fig. S1). The car-
boxyl-terminal part of α14 completes an antiparallel four-helix
bundle with α15, α16, and α17 of D4. Helix α17 projects away
from the bundle and forms a loose hairpin with α18.
Conserved Surface-Located Residues Within CagA Point Toward Four
Functional Areas. Mapping of the surface-exposed invariant and
conserved residues to CagA1–884 structure reveals a clustering into
four main conserved surface-exposed patches (CSP1–4) (Figs. 2
and 3A). CSP1 is located in helix α18 and the C-terminal portion
of helix α17. These helices self-interact with a symmetry-related
subunit in a head-to-tail manner (buried surface of 543Å2) (Fig.
S4A). CagA was found to form multimers via the MKI sequences
(also called CagA multimerization sequence-CM) and this mul-
timerization increase CagA activity on the SHP2 phosphatase (20,
21). Because the MKI (CM) sequences are not present in the
CagA1–884 fragment, the oligomeric state of CagA1–884 was inves-
tigated. Size-exclusion chromatography coupled to multiangle light
scattering shows that CagA1–884 is a monomer in solution (Fig.
S4B), suggesting that the observed dimerization is likely a result
of crystal packing. However, given the position and conservation
of the residues present at this interface, this region might rep-
resent a dimerization zone in the context of the full-length CagA
or form a possible hot spot for protein–protein interactions.
A second conserved surface patch, CSP2, is located in the distal
part of the helical bundle formed by the carboxyl-terminal portion
of α14, α15, α16, and α17 (Fig. 3A). This left-handed helical
bundle shows similarities with the F-actin binding domain of the
Bcr-Abl tyrosine kinase (rmsd 2.4), but also with cytoskeleton
proteins such as α-catenin (rmsd 2.7), vinculin (rmsd 2.5), and the
focal adhesion kinase (FAK, rmsd 2.2) (Fig. S5). A remarkable
and key property of CagA is that it can modify several types of
cell-to cell junctions (22). In particular, CagA colocalizes with and
affects FAK and vinculin phosphorylation at FAs (7, 23, 24).
CagA also disrupts the E-cadherin/β-catenin complex at adherens
junctions (8, 9). The structural similarity and sequence conser-
vation suggests that the CagA helical bundle might be important
for interaction of CagA, with some of these membrane proteins
possibly by mimicry of the F-actin binding domain motif.
A third area of interest, CSP3, consists of a patch of basic
residues belonging to α13 of the D2′′ domain. CSP3 includes
Fig. 3. Potential functional sites in CagA structure. (A) Semitransparent
surface representation of the CagA domain D3-D4 (Left) and D2 (Right)
colored in gray with strictly conserved and conserved residues colored in red
and pink, respectively. The location of the conserved surface exposed patch
“CSP1–4” are indicated. (B) Surface representation of α12 and α13 colored by
electrostatic potential. Residues forming the basic patch potentially involved
in PS binding are indicated.
14642 | www.pnas.org/cgi/doi/10.1073/pnas.1206098109
residues R624 and R626, which were previously found to interact
with phosphatidylserine (PS) (25) (Fig. 3). The structure reveals
that additional basic residues of D2′′, such as K613, K614, K617,
and K621 might also contribute to PS binding (Fig. 3B). The
fourth conserved area CSP4 is discussed below.
Interaction Between CagA SLB and β1 Integrin Is Required for
Translocation. The N-terminal half of the CagA protein (amino
acids 1–612, CagA1–612) containing domains D1 and most of D2
has previously been shown to interact with α5β1 integrin (6). The
binding of α5β1 integrin by CagA is believed to be a prerequisite
for CagA internalization into the host cells. To further narrow
down the β1 integrin-binding domain within CagA1–612, we per-
formed a yeast two-hybrid (YTH) protein-interaction screen.
The CagA1–612 insert in the YTH bait vector was systematically
shortened from both ends to generate eight subclones (Y31–
Y38), producing recombinant CagA proteins differing in length
by 100 aa each (Fig. 4A). The YTH screen revealed interaction
of β1 integrin with three CagA subclones (Y31, Y35, Y36), all of
which shared the CagA303–404 amino acid sequence (Fig. 4A). In
the crystal structure, these residues correspond to the first five
strands of the SLB and part of the D2′ domain (Fig. 1B). This
area also coincides with the fourth conserved surface patch,
CSP4, found in the CagA structure (Figs. 2 and 3A).
To corroborate the specific interaction of this region with
β1 integrin, competition experiments were performed using
recombinant CagA303–404 (Fig. 4 A and B). CagA303–404 identi-
fied in the YTH screen was expected to compete with native
CagA binding of H. pylori and eventually to interfere with CagA
translocation into gastric adenocarcinoma (AGS) cells. The cag-
PAI+ H. pylori strain P12 translocates CagA into AGS cells, as
verified by its EPIYA motif tyrosine phosphorylation (Fig. 4B,
lane 1). Using the read-out of CagA phosphorylation, the
purified CagA303–404 strongly inhibited CagA translocation in a
dose-dependent manner (Fig. 4B). However, the CagA303–404
fragment was not very stable in solution, likely because it encom-
passs only part of the D2′ domain and thus lacked structural in-
tegrity. To determine whether the SLB or D2′ were involved in β1
integrin binding, we generated and purified CagA recombinant
fragments based on the crystal structure (Fig. 1A) (CagA303–456,
CagA369–448) and competition experiments were performed (Fig.
4C). Binding to AGS cells was detected for all these proteins, but
not with the same affinity (Fig. 4C) (α-His). Although the same
amount of proteins were incubated, CagA1–273, CagA1–291 (cov-
ering D1), CagA303–456 (proximal part of the SLB and D2′), and
CagA392–733 (containing the distal part of the SLB and D2′′)
were found to interact more strongly with AGS cells than
CagA369–448 (containing only D2′). The binding of the fragment
CagA392–733 on AGS cells might be attributed to the presence of
the PS-binding patch at its surface, because PS is externalized
during H. pylori infection (25). The CagA translocation assay
shows that the purified CagA303–456 fragment inhibited CagA
translocation by H. pylori (Fig. 4C). In the same assay, neither
CagA1–273, CagA1–291, CagA369–448, nor CagA392–733 showed any
inhibitory effect on CagA translocation (Fig. 4C). These results
indicate that the first five strands of the SLB (residues 303–368)
are sufficient to inhibit CagA translocation in our assay. Recom-
binant Yersinia invasin (GST-Inv397), which binds β1 integrin via
a RGD (arginyl-glycyl-aspartic acid)-like motif, did not interfere
either with CagA translocation (Fig. 4C). Taken together with the
YTH interaction data, these functional assays show that the
proximal part of the SLB of CagA binds to β1 integrin and that
this interaction is an essential step of CagA translocation.
Discussion
CagA is a remarkably versatile oncoprotein produced by H.
pylori, known to interact with a plethora of host signaling fac-
tors and to promote gastric cancer development. The structure
Kaplan-Türköz et al.

of CagA1–884 presented here provides a clear definition of the
domain organization of the large, stable N-terminal region of
CagA, covering about two-thirds of the whole CagA protein.
Some of these domains share different degrees of similarities
with prokaryotic or eukaryotic proteins, the aim of which might
be protein mimicry, which could explain how different domains
of CagA can affect location of the protein in H. pylori as well as
the eukaryotic target cell (26–29). Another remarkable char-
acteristic of CagA N-terminal region is its flexibility, in par-
ticular the domain D1 (residues 1–300). Previously, most of the
C-terminal region of CagA was found disordered, even when
bound to the Par1b/MARK2 kinase (14). Here we found that
domain D1, as well a number of loops of D2, D3, and D4, are
highly flexible. This flexibility could be attenuated in the
context of the full-length protein, because the N terminus was
found to interact with the C-terminal domain (28). Neverthe-
less, both in vitro and in vivo proteolysis studies showed
that the protein is rather unstable and heavily prone to deg-
radation (10, 11). This intrinsic flexibility might play an im-
portant role for CagA to accommodate interactions with
multiple partners.
Mapping of conserved residues within the CagA structure
reveals four conserved surface patches (CSP1–4) that represent
candidate hot spots within CagA for the interaction with host-cell
molecules. CSP3 contains residues that were shown to be involved
in the binding to PS. The binding of CagA CSP3 might contribute
to tether CagA at the inner leaflet membrane, where PS is more
abundant (25). Another important area is CSP4, which was shown
to constitute the binding interface with α5β1 integrin. We provide
clear evidence that this interaction is required for internalization
of CagA into the host cell. In the CagA structure, these residues
are part of the SLB, but also of the β6β7 hairpin (Fig. 4D) and
form a large hydrophobic cleft that represents a large interaction
interface for integrin (Fig. 4E). Previous quantification of the
CagA/integrin binding by plasmon resonance studies indicated
that α5β1 integrin binds CagA approximately 100-fold stronger
than its natural ligand, fibronectin (KD of 0.15 nM vs. 15 nM,
respectively) (6). In this respect, we note that recombinant Yer-
sinia invasin (GST-Inv397) did not interfere with CagA trans-
location (Fig. 4C). Invasin is a well-known bacterial virulence
factor that binds with high affinity to α5β1 integrin to mediate
efficient and rapid internalization of the bacteria into host cells,
especially M cells in the gut (30). The structure of invasin mimics
Kaplan-Türköz et al.
Fig. 4. Mapping the CagA-β1 integrin interaction
domains. (A) Schematic representation of CagA frag-
ments interacting with β1 integrin in a YTH screen.
Construction of systematically shortened CagA1–612
clones (Y31–Y38) in the bait vector (gray represents
deleted sequences). The green square denotes the CagA
amino acid sequence essential for interaction of CagA
with β1 integrin. Clones Y32 and Y34 grew on selective
medium without interaction partner (autoactivation).
(B) Recombinant CagA303–404 interferes with CagA
translocation. AGS cells were incubated with 4, 8, or 12
nMol purified CagA303–404 (lanes 2–4) and infected with
H. pylori P12 or not (lane 5). Lysates were run on SDS/
PAGE, blotted, and reacted with antibodies as in-
dicated. (C) AGS cells were incubated with purified
CagA fragments (His-tagged; see Fig. 1B for their exact
location) or recombinant Yersinia invasin (GST-Inv397),
and the structure-derived fragments CagA303–456 or
CagA369–448 and infected with H. pylori P12 strain.
Lysates were run on SDS/PAGE or single gel system,
blotted, and reacted with antibodies, as indicated. (D)
Ribbon diagram of CagA1–884 domain D2 (gray) showing
the β1 integrin interacting portion (magenta). (E) Sur-
face representation of the structure in the same orien-
tation colored according to the electrostatic potential.
the RGD motif of fibronectin to bind to β1 integrin (31). CagA
does not contain a RGD motif, meaning that CagA binding to β1
integrin is via a different, so far unexplored mechanism, probably
by binding to a different domain of β1 compared with invasin.
Another H. pylori protein, CagL, was originally reported to bind
to β1 integrin via a RGD motif (7), whereas other reports showed
a RGD-independent binding of CagL to β1 integrin (6). However,
because invasin did not impair CagA translocation, our work
indicates that the RGD binding motif of β1 integrin is not re-
quired for CagA translocation, suggesting that CagA binds to
a different region within β1 integrin.
The competitive binding of the SLB fragments to β1 integrin
heterodimers on the surface of gastric AGS cells suggests that it
functionally interferes with binding of full-length CagA during H.
pylori infection. Which domain of β1 integrin is bound by CagA is
presently unknown. The SLB of CagA, in particular the β1-
integrin binding domain, revealed an interesting structural sim-
ilarity with the outer surface protein OspA of B. burgdorferi.
These bacteria cause Lyme disease and use outer surface pro-
teins to adhere to human cells once transmitted by tick bites
(reviewed in ref. 32). Although an interaction of OspA with β1-
integrin receptors has not been described, a direct interaction of
OspA and OspB with the complement receptor 3 (CR3) has
been reported (33). CR3 is a αMβ2 integrin heterodimer spe-
cifically expressed by immune cells, such as neutrophils, dendritic
cells, and B or T cells, and is required for phagocytosis of B.
burgdorferi into human monocytes (34). Therefore, it might be
speculated that OspA could interact with β2 integrins via its SLB,
similarly to the way CagA interacts with β1 integrin. Whether
CagA binds β2 integrins through this conserved surfaced as well
has yet to be determined.
Why does CagA, which is the translocated effector protein and
not necessarily a structural component of the cag-T4SS, interact
with the integrin receptor? The question whether the CagA/β1
integrin interaction is an epiphenomenon or is essential for
CagA translocation cannot be studied by gene deletion, because
CagA is the only effector protein of the cag-T4SS. However, the
effective blocking of CagA translocation by CagA SLB fragments
MICROBIOLOGY
provided by our study clearly proves that the specific interaction
of CagA with the integrin receptor is an essential step for the
translocation process of CagA into the host cell. A complete
picture about the intricate interaction of the cag-T4SS and the
β1-integrin receptor will probably be obtained when the precise
PNAS | September 4, 2012 | vol. 109 | no. 36 | 14643
binding domains within the integrin, as well as the binding of the Table 2. Phasing and refinement statistics
other components, CagI, CagL, and CagY to the integrin re- Phasing and refinement statistics
ceptor have been determined. Besides being important for
translocation, the CagA N-terminal domain also interacts with Phasing
intracellular partners such as ASPP2 (16), RUNX3 (17), TAK1, No. of sites 8
and TRAF6 (18). The structure of CagA1–884 we describe here Phasing power 1.2
will enable a more in-depth functional characterization of CagA Figure of merit (FOM) 0.27
interactions with these proteins to understand how it promotes FOMdm (59% solvent) 0.57
carcinogenesis, and marks an important first step toward the Refinement
development of specific inhibitors. Resolution range for refinement (Å) 62.7–3.6
No. of unique reflections in refinement 13,349
Materials and Methods Working set 12,679
Cloning, Mutagenesis, Expression, and Purification of CagA1–884, Se-CagA1–884, Test set 670
and Se-CagA1–884 Mutants. The DNA fragment encoding amino acid residues Completeness (%) 91.8
1–884 of CagA (CagA1–884) from H. pylori strain 26695 was cloned into the Monomers/ASU 1
pET101D topo vector (Invitrogen). Single-point mutations (L699M and No. of protein atoms 3,283
L727M) were introduced using the site-directed mutagenesis kit (Stra- Rwork/Rfree 0.3287/0.3413
tagene). BL21 Star (DE3) cells carrying the resulting plasmids were grown
Rmsd bonds (Å) 0.002
in LB media containing ampicillin at 37 °C until an OD600 of 0.7 and protein
Rmsd angles (°) 0.593
expression was induced by the addition of 1 mM isfopropyl-β-D-thio-
galactopyranoside and cells were grown overnight at 20 °C. Briefly, proteins Mol Probity score 2.2
were purified using immobilized metal affinity chromatography, anion ex- Ramachandran plot
change, and gel-filtration chromatography. Selenomethionine-substituted Most favored (%) 95.8
CagA1–884 was obtained following the protocol previously described (35). Allowed (%) and generously allowed (%) 4.0
Se-CagA1–884, Se-CagA1–884 L699M, and Se-CagA1–884 L727M mutants were Outliers (%) 0.2
produced and purified as native except that 1 mM DTT was added in
all buffers.

Crystallization, Structure Determination, and Refinement. CagA1–884, Se- 59% (37). Phasing statistics are given in Table 2. The experimental map
CagA1–884, Se-CagA1–884 L699M, and Se-CagA1–884 L727M crystals were was of excellent quality (Fig. S6) and the model was built using COOT
obtained by using the vapor-diffusion methods in hanging drops. One mi- (40). The atomic positions and TLS (translation, libration, screw)
croliter of protein was mixed with 1.0 μL of reservoir solution containing parameters refined using BUSTER (41) and PHENIX (42) (Tables 1 and 2). The
ethanol 10–14%, (vol/vol), 100 mM sodium cacodylate pH 6–7, and sodium final model includes residues 319–343, 349–478, 489–510, 536–641, 644–
iodide 15 mM. Crystals of 200 μm × 200 μm × 70 μm appeared in 3 to 4 d at 706, 718–736, 738–758, and 764–822. The structure has been deposited in
20 °C and were grown during 2 to 3 wk. Crystals were flash-frozen in liquid the Protein Data Bank (PDB ID code: 4GOH). Figures in the article were
nitrogen after step-wise addition of cryoprotectant solution consisting of generated using Pymol (43).
20% (vol/vol) ethanol, 100 mM sodium cacodylate, 15 mM sodium iodide,
and 18% ethylene glycol. X-ray diffraction data were collected from native Bacterial Strains, Cell Lines, and Culture Conditions. The H. pylori cag-patho-
and selenomethionine derivative crystals at the ID29 beamline of the Eu- genicity island-positive strain P12 was used for infection experiments (44).
ropean Synchrotron Radiation Facility (Grenoble) or at SOLEIL (CagA1–884 H. pylori was grown on GC agar plates (Difco), as described previously (44).
mutants). Crystals belonged to the tetragonal space group P41212 with unit The human gastric cell line AGS (CRL-1739) was used for infection experi-
cell dimension of a = b = 98 Å and c = 244 Å, and diffracted to a resolution ments. Cells were grown in RPMI containing 10% FCS, at 5% CO2, and 37 °C
of 3.6 Å (Native) and 3.9 Å (SAD) (Tables 1 and 2). The diffraction data were and subcultured every 2–3 d.
indexed and integrated using XDS (36) and scaled with SCALA from the CCP4
program suite (37). Data collection statistics are given in Table 1. The YTH Assay. The Invitrogen system consists of the entry vector pDONR207, and
structure of CagA1–884 was solved by the SAD method using selenomethio- destination vectors pGADT7 (prey vector) and pGBKT7 (bait vector). YTH bait
nine derivative data. The position of Se atoms was determined with SHELXD and prey libraries were generated comprising gene sequences covering the
(38), phases calculated with the program SHARP (39), and density modifi- complete external β1-integrin region. For plasmid cloning, the recombina-
cation was performed using SOLOMON and DM with a solvent content of tory cloning procedure, as described by the manufacturer, was used. Bait
and prey plasmids were transformed into the haploid Saccharomyces cer-
evisiae strains Y187 and AH109. Diploid yeast cells were selected after
Table 1. Data collection statistics mating and selection on SD medium lacking tryptophan (Trp) and leucine
(Leu), thus generating all possible combinations of bait and prey plasmids.
Data collection Se-Met Native
After growth on SD-Leu/Trp medium, yeast colonies were transferred to SD-
Space group P41212 P41212 Leu/Trp/His medium to select for interactions. Growth after 3–6 d indicated
Wavelength (Å) 0.97903 0.97627 bait–prey interactions. Additionally, the stringency of this screen was en-
hanced by selection on SD-Leu/Trp/His medium containing the competitive
Cell parameters, 98.2, 98.2, 243.15 97.8, 97.8, 245.4
inhibitor 3-aminotriazole (5 mM).
a, b, c (Å)
Resolution 98.2–3.90 (4.11–3.90) 90.87–3.60 (3.79–3.60)
Antibodies and Reagents. Antibodies against phosphotyrosine were obtained
range (Å) from Santa Cruz (PY99) or Upstate (4G10), and anti-His antibody from
Rmerge 0.07 (0.70) 0.07 (0.85) Genescript. Polyclonal HRP and alkaline phosphatase-conjugated anti-mouse
Rp.i.m. 0.044 (0.44) 0.059 (0.68) IgG and anti-rabbit IgG antisera, HRP-conjugated streptavidin, protease
No. of observed 72,769 (10,315) 46,117 (7,097) inhibitors PMSF, leupeptin, and pepstatin were obtained from Sigma. AK257
reflections and AK268 are rabbit polyclonal antisera against C-terminal or N-terminal
No. of unique 11,530 (1,638) 13,408 (1,974) CagA, respectively, actin-HRP and mouse anti-HIS (GenScript), Integrin-β1
reflections clone LM534 (Chemicon), anti-GST (Sigma).
Mean [I/s(I)] 10.1 (2.2) 6.2 (1.4)
Completeness (%) 99.9 (99.9) 92.8 (96.4) CagA Translocation Assay (Tyrosine Phosphorylation of CagA). Gastric AGS cells
Multiplicity 6.3 (6.3) 3.4 (3.6) were infected with H. pylori at 70–90% confluency with a multiplicity of
infection of 60. For competitive inhibition experiments, recombinant purified
Values in parentheses refer to the indicated resolution shell. Se-Met, sele- CagA proteins (11–19 nmol/1 × 106 cells), or recombinant Yersinia invasin
nomethionine. known to bind to β1 integrin (GST-Inv397, 50 μg/1 × 106 cells, control), were

14644 | www.pnas.org/cgi/doi/10.1073/pnas.1206098109 Kaplan-Türköz et al.

added to the cells (4 °C, 30 min) before the infection with H. pylori. After 3 h
of infection, cells were harvested in PBS with protease inhibitors (pepstatin
1 μM, leupeptin 1 μM, PMSF 1 mM) and phosphatase inhibitor sodium van-
adate (1 mM). Harvested cells were centrifuged (500 × g, 5 min, 4 °C) and
pellets lysed in RIPA buffer with protease and phosphatase inhibitors and
DNase I for later SDS/PAGE or single-gel system (45) in nonreducing, denaturing
conditions, and immunoblot analysis. See SI Materials and Methods for further
details of methods used, and Fig. S7 for the structures of CagA1–884 mutants
contoured with the anomalous difference Fourrier electron density maps.
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ATIP-Avenir Program from Centre National de la Recherche Scientifique and
Ligue Contre le Cancer and by Deutsche Forschungsgemeinschaft SFB914
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