Identification of Chromosomal HP0892-HP0893
Toxin-Antitoxin Proteins in Helicobacter pylori and Structural
Elucidation of Their Protein-Protein Interaction*
Kyung-Doo Han‡, Do-Hwan Ahn§, Seung-A Lee§, Yu-Hong Min¶, Ae-Ran Kwon¶, Hee-Chul Ahn?, and Bong-Jin Lee§1
From the‡Advanced Analysis Center, Korea Institute of Science and Technology, Seoungbuk-gu, Seoul 136-791, Korea, the
§Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Gwanak-gu, Seoul 151-742,
Korea, the¶Department of Herbal Skin Care, Daegu Haany University, Gyeongsan, Gyeongsangbuk-do 712-715, Korea, and
the?Department of Pharmacy, College of Pharmacy, Dongguk University, Goyang, Geonggi 410-820, Korea
Background: HP0892 of H. pylori shares high structural similarity with other toxin-antitoxin (TA) system toxins.
Results: RNase activity and cell toxicity of HP0892 is inhibited by HP0893 via strong protein-protein interaction.
Conclusion: HP0892-HP0893 pair is a TA system with a protein-protein interaction mode similar to other TA pairs.
Significance: It is highly probable that HP0892-HP0893 TA pair is involved in H. pylori virulence.
Bacterial chromosomal toxin-antitoxin (TA) systems have
been proposed not only to play an important role in the stress
response, but also to be associated with antibiotic resistance.
Here, we identified the chromosomal HP0892-HP0893 TA
proteins in the gastric pathogen, Helicobacter pylori, and
structurally characterized their protein-protein interaction.
toxin based on its structural similarity to other RelE family
TA toxins. In this study, we demonstrated that HP0892 binds
to HP0893 strongly with a stoichiometry of 1:1, and HP0892-
HP0893 interaction occurs mainly between the N-terminal
secondary structure elements of HP0892 and the C-terminal
region of HP0893. HP0892 cleaved mRNA in vitro, preferen-
was inhibited by HP0893. In addition, heterologous expres-
sion of HP0892 in Escherichia coli cells led to cell growth
arrest, and the cell toxicity of HP0892 was neutralized by
co-expression with HP0893. From these results and a struc-
tural comparison with other TA toxins, it is concluded that
HP0892 is a toxin with intrinsic RNase activity and HP0893 is
an antitoxin against HP0892 from a TA system of H. pylori. It
has been known that hp0893 gene and another TA antitoxin
gene, hp0895, of H. pylori, are both genomic open reading
frames that correspond to genes that are potentially
expressed in response to interactions with the human gastric
pylori are involved in virulence of H. pylori.
Programmed cell death (PCD)2refers to any form of cell
tems such as apoptosis (2) or autophagy (3) in eukaryotic mul-
ticellular organisms play significant roles in a variety of biolog-
ical processes including development, cell homeostasis, and
oncogenesis (4). Traditionally, PCD has been considered asso-
ciated with only eukaryotic multicellular organisms; however,
eubacteria and archaea, the most common mechanism
TA systems are usually composed of a stable toxin that
arrests cell growth and a labile antitoxin that counteracts the
toxin. The former always exists as a stable protein, whereas the
latter is either a protein (type II TA system) or an RNA species
(type I or III TA system) (6). In the type I TA system, the anti-
toxins are small antisense RNAs forming duplexes with the
toxin mRNAs, which repress translation of the toxin genes (7).
In the type II system, the antitoxins are proteins that neutralize
the toxins by a direct protein-protein interaction with the tox-
ins (8). In type III TA systems, small RNA antitoxin combines
with and neutralizes protein toxins (9, 10). In most cases, toxin
and antitoxin genes are in an operon where the antitoxin gene
often locates upstream of the toxin gene (6), usually overlap-
usually negatively autoregulated at the transcription level by
antitoxins and TA complexes, which bind to the TA locus pro-
Several TA systems have been discovered in bacterial plas-
mids and on chromosomes. Plasmid-borne TA systems are
do not inherit a copy of a plasmid upon division are killed
stable toxin. On the contrary, the biological roles of chromo-
* This work was supported by National Research Foundation of Korea
Grants 20110001207 and 2012R1A2A1A01003569 funded by the
Ministry for Health, Welfare, and Family Affairs, Republic of Korea
Grants A084420 and A092006; 2011 BK21 Project for Medicine, Den-
tistry, and Pharmacy; and Korea Institute of Science and Technology
Author’s Choice—Final version full access.
1To whom correspondence should be addressed. Tel.: 82-2-880-7869; Fax:
82-2-872-3632; E-mail: firstname.lastname@example.org.
Bank; TA, toxin-antitoxin; trHNCA, TROSY-type HNCA; TROSY, transverse
relaxation optimized spectroscopy.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 8, pp. 6004–6013, February 22, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
Published in the U.S.A.
somal TA systems have been the subject of widespread discus-
sion and speculation. Recently, nine possible functions includ-
ing genomic junk, stabilization of genomic parasites, selfish
alleles, gene regulation, growth control, and production of per-
cell to stress conditions such as starvation is regarded to be the
major function of chromosomal TA systems. Moreover, TA
systems can contribute to the formation of persistent cells dur-
ing exposure to antibiotics, and recently, persistence was sug-
gested as a common function of TA systems (13, 14).
The Escherichia coli K12 chromosome possesses at least
eight relatively well characterized TA systems: MazF-MazE,
RelE-RelB, ChpBK-ChpBI, YafQ-DinJ, YoeB-YefM, HipA-
HipB, YafO-YafN, and MqsR-MqsA (6). Among the toxins of
these modules, the RelE, YafQ, and YoeB toxins are classified
within the same family (RelE family) due to low but significant
inhibiting translation through mRNA cleavage (8). Among
those toxins, RelE is a ribosome-dependent endoribonuclease
that is only active when associating with a ribosome (15–17),
whereas the others have intrinsic ribonuclease (RNase) activity
and are regarded to function as ribosome-dependent mRNA
interferases, too (6, 19, 20).
Helicobacter pylori is a Gram-negative neutralophile that
to survive in an acidic environment of human stomach and to
environment of the stomach, it has developed acid resistance
mechanisms (21). For example, urease of H. pylori, which has
optimum activity at neutral pH, produces NH3from gastric
juice urea, and neutralizes acidity around the bacterium (21).
Because of these acid resistance mechanisms, H. pylori is
thought to possess an intracellular pH near neutrality even in
associated lymphoid tissue lymphoma, and gastric cancer (25–
27). H. pylori is one of the most common chronic bacterial
infections of humans, infecting more than half of the world
In our previous study (28), we found that HP0894 (a YafQ-
teins of H. pylori 26695 strain form a TA system. In another
study of ours (29), we reported the solution structure of the
conserved hypothetical protein HP0892 from this strain and
found that its structure is very similar to those of HP0894 and
other RelE family TA toxins. Those findings about HP0892
prompted us to speculate that HP0892 might also act as a TA
toxin with intrinsic RNase activity like HP0894. In this study,
we identified the biological function of HP0892 as a TA toxin
previously for HP0892 (29). The same procedure was used to
prepare the cells harboring the recombinant plasmids for
mutated HP0892. All resulting HP0892 constructs contained
eight non-native residues (LEHHHHHH) at the C terminus
(referred to as HP0892-His). Those residues facilitated subse-
quent protein purification. Additionally, the same procedure
with minor change was used for HP0892 without any purifica-
tion tag or non-native residues (in vector pET-21a; Novagen,
Madison, WI) and for glutathione S-transferase (GST) fusion
HP0893 (referred to as GST-HP0893) (in vector pGEX-4T-1;
GE Healthcare) and HP0893 with 20 non-native residues
including a His tag at the N terminus (referred to as His-
HP0893) (in vector pET-28a; Novagen).
out as described previously (29) for HP0892-His. For the pro-
duction of15N- or15N,13C-labeled proteins, M9 medium con-
taining15NH4Cl and/or [13C]glucose as stable isotope sources
were used. The protein samples were purified using a Ni2?-
affinity column following standard protocol. The same proce-
dures were used to prepare His-HP0893 and mutated HP0892-
His. For His-HP0893, because it is a putative DNA-binding
protein, further purification using polyethyleneimine was car-
ried out. Polyethyleneimine was added to the previously puri-
fied His-HP0893 solution at a final concentration of 0.08%,
tion, the supernatant was collected, dialyzed against a 50 mM
borate buffer (pH 11), and loaded onto a disposable PD-10 col-
umn (GE Healthcare) packed with Q-Sepharose fast flow resin
(GE Healthcare). The column was washed with 100 ml of the
same buffer, and then the protein was eluted with a 50 mM
borate buffer (pH 11) containing 1 M NaCl.
Construction of HP0892/HP0893 Co-expression Systems—
The full-length hp0892 and hp0893 genes were amplified from
H. pylori genomic DNA by PCR as different DNA fragments
and were subsequently subcloned under the T7 promoter in
pET-21a (ampicillin-resistant) and pET-29a (kanamycin-resis-
tant) vectors (Novagen), respectively. These two vectors were
co-transformed into E. coli BL21 (DE3) (HP0892/HP0893 co-
expression system I). In this case, the HP0893 construct con-
tained eight non-native residues including a His tag at the C
terminus (referred to as HP0893-His). The HP0892 construct
contained no additional residues. Using the same procedure,
another co-expression system composed of HP0892 (in vector
pET-21a) and HP0893 (in pET-28a) was constructed (HP0892/
struct contained 20 non-native residues including a His tag at
the N terminus (referred to as His-HP0893). The HP0892 con-
struct contained no additional residues.
Pulldown Assay for HP0892/HP0893 Binding—E. coli BL21
21a) were cultured and expressed with 0.5 mM isopropyl-?-D-
thiogalactopyranoside, separately. The cells were harvested by
centrifugation and resuspended with cell lysis buffer (20 mM
sodium phosphate buffer (pH 7.3)), separately. The two cell
cultures were mixed and disrupted by sonication. The lysate
was centrifuged at 10,000 rpm, 4 °C for 1 h, and the superna-
umn was washed with the same cell lysis buffer until the UV
peak stabilized. The adsorbed protein was eluted with a gluta-
thione buffer (3.08 g of reduced glutathione in 1 liter of 50 mM
Tris-HCl buffer (pH 8.0)). The eluted solution was analyzed
with sodium dodecyl sulfate-polyacrylamide gel electrophore-
sis (SDS-PAGE). The fractions containing the target proteins
were pooled, and buffer exchange was carried out with 20 mM
Tris-HCl buffer (pH 7.9) containing 0.5 M NaCl. The solution
with the 20 mM Tris-HCl buffer (pH 7.9) containing 60 mM
imidazole and 0.5 M NaCl. The adsorbed proteins were eluted
lyzed by SDS-PAGE.
Co-expression and Co-purification of HP0892/HP0893—
HP0892 and HP0893 were co-expressed and co-purified using
the HP0892/HP0893 co-expression system I described above.
(ampicillin 50 ?g/ml and kanamycin 40 ?g/ml). Induction, cell
harvest, cell lysis, and purification with a Ni2?-affinity column
were done following standard protocols. The fractions eluted
ent) were analyzed by SDS-PAGE.
Toxicity of HP0892 in E. coli—E. coli BL21 (DE3) cells har-
boring the HP0892 expression plasmids (HP0892/pET21a)
and the HP0892/HP0893 co-expression plasmids (HP0892/
of LB broth containing antibiotics (50 ?g/ml ampicillin for
pET21a and HP0892/pET21a, and 50 ?g/ml ampicillin and 40
?g/ml kanamycin for HP0892/HP0893 co-expression system
II). At an A600of approximately 0.3, 1 mM isopropyl-?-D-thio-
galactopyranoside was added. Subsequently, samples for viable
counts and SDS-PAGE analyses were taken at several time
points. Viable counts were made by plating dilutions of the
cultures onto LB plates containing the appropriate antibiotics.
at each time point were analyzed by SDS-PAGE.
In Vitro RNA Cleavage by HP0892—A DNA fragment con-
taining the T7 promoter and the hp0893 gene was obtained by
PCR amplification of H. pylori genomic DNA. The hp0893
mRNA was synthesized in vitro from this DNA fragment using
the T7-MEGAshortscript kit (Ambion, Austin, TX). Mixtures
of hp0893 mRNA (?310 bases, 300 ng), HP0892, HP0892
mutants, and/or HP0893 were prepared in several ratios in 20
mM Tris-HCl (pH 7.4) buffer containing 150 mM NaCl (total
volume, 5 ?l). The reaction was done at 37 °C for 60 min and
was stopped by adding 5 ?l of the sequencing loading buffer
ing ethidium bromide with Tris borate/EDTA buffer.
Primer Extension—The hp0893 mRNA was used as a primer
template. Two different DNA primers were synthesized and
5?-labeled with [?-32P]ATP using T4 polynucleotide kinase
(New England Biolabs). Primers (0.8 pmol) were annealed to
each template (1 pmol) in a 10-?l mixture samples by incuba-
tion at 90 °C for 5 min followed by slow cooling to room tem-
perature. The reaction mixture was digested by adding 1 ?l of
(250 mM Tris-HCl (pH 8.3), 375 mM KCl, and 15 mM MgCl2), 5
transcriptase (Invitrogen) for a final reaction volume of 20 ?l.
The cDNA was synthesized at 55 °C for 30 min and then puri-
fied by phenol/chloroform extraction and ethanol precipita-
and analyzed by autoradiography.
against HP0892, a 30-residue peptide corresponding to the
C-terminal region (Glu-66 to Leu-95) of HP0893 was commer-
cially synthesized (Anygen, Kwangju, Korea) and purified (96%
purity). The two-dimensional1H,15N TROSY spectra of15N-
labeled HP0892 alone (final concentration 0.1 mM) and with
HP0893Ctp added incrementally (0.05, 0.1, and 0.2 mM) to the
sample were obtained on a Bruker Avance 600 spectrometer.
The averaged chemical shift changes were calculated by (30).
??ave? ????HN?2? ???N/6.57?2?0.5
HP0893Ctp, the three-dimensional TROSY-type HNCA
(trHNCA) of13C,15N-labeled HP0892 (0.2 mM) mixed with
HP0893Ctp (0.3 mM) was obtained on a Varian VNMRS 900
spectrometer. All NMR samples were dissolved in 20 mM
sodium phosphate (pH 6.0) buffer without any additional salt.
The NMR samples contained 10% D2O for the lock signal. All
spectra were processed and analyzed using NMRPipe/
NMRDraw (31) and NMRView (32), respectively.
Gel Filtration Chromatography—A mixture of HP0892 with
HP0893 was prepared by co-lysis of cells harboring HP0892-
His and His-HP0893, respectively, and subsequently, Ni2?-af-
mass markers were fractionated on a Superdex 75, 10/300 GL
column (GE Healthcare) using an AKTA FPLC system (GE
Healthcare). The mobile phase consisted of 50 mM sodium
phosphate (pH 7.4) buffer containing 100 mM NaCl. The void
volume was measured with blue dextran 2000 (2000 kDa). The
proteins were eluted at flow rates of 0.5 ml/min and were mon-
itored using UV light at 280 nm.
the toxin gene in TA systems. On the H. pylori chromosome,
the hp0893 gene is located directly upstream of the hp0892
gene (Fig. 1). Although HP0893 has been known to be a hypo-
thetical protein, we expected that if HP0892 is a TA toxin, then
pylori. To identify the potential interaction between HP0892
and HP0893, samples of protein mixtures including HP0893
fused with GST (GST-HP0893) and HP0892 fused with His tag
at the C terminus (HP0892-His) were purified by the pulldown
FIGURE 1. Structure of the region of the H. pylori chromosome encoding
hp0892 and hp0893.
one-Sepharose and subsequently, Ni2?-affinity column. As
of both proteins, HP0892-His and GST-HP0893, in the same
trol, we also monitored the interaction between HP0892-His
and GST alone by the same pulldown assay method, but no
interaction was observed (data not shown). To confirm the
interaction between HP0892 and HP0893, both proteins were
(see “Experimental Procedures”) and then co-purified. For
detection and purification, HP0893 was expressed as a C-ter-
minal His tag fusion (HP0893-His) and HP0892 was expressed
imidazole treatments revealed the presence of both expressed
proteins (HP0893-His and HP0892) in the same eluted frac-
tions from the Ni2?-affinity column (Fig. 2B). These results
indicate that there is a stable and strong interaction between
the HP0892 and HP0893 proteins.
Inhibitory Effect of HP0892 on E. coli Cell Growth Is Neutral-
ized by HP0893—The potential of HP0892 as a toxin and of
HP0893 as an antitoxin was examined by measuring their
effects on the viability of E. coli cells grown in the presence of
isopropyl-?-D-thiogalactopyranoside. As shown in Fig. 3, the
number of viable E. coli cells decreased significantly with
expression of HP0892, but co-expression of HP0893 neutral-
ized the detrimental effect of HP0892 on E. coli cell growth.
In Vitro RNase Activity of HP0892 Is Inhibited by HP0893—
Todetermine whether HP0892 has intrinsic RNase activity, we
tested the in vitro RNase activity of HP0892 and the inhibitory
H. pylori is very likely to possess an intracellular pH near neu-
trality in even acidic environment of stomach, so this test and
pH condition (pH 7.4). As shown in Fig. 4, in vitro synthesized
mRNA was digested into smaller fragments following incuba-
tion with HP0892, whereas addition of HP0893 inhibited
mRNA decay by HP0892. HP0893 alone did not digest mRNA.
In addition, the RNase activity of a single mutant HP0892
HP0892 was not due to contamination of RNases during puri-
fication and that the His-86 residue is a key catalytic residue of
Primer Extension—To identify the sequence of HP0892
cleavage site on mRNA, HP0892-mediated cleavage of hp0893
mRNA was analyzed by primer extension experiments. As
adenine (A) or guanine (G) residues, although not all of A or G,
and AG-rich sequence regions were relatively more sensitive.
Binding Sites of HP0892 and HP0893—Because HP0893
exists as an aggregated form with a high molecular mass and is
HP0893 and could not investigate the binding aspect between
HP0893 protein sample. In the H. pylori HP0894-HP0895 TA
toxin is responsible for binding with the HP0894 toxin (28),
which is a structural and sequential homologue close to
HP0892. In addition, in E. coli YoeB-YefM and RelE-RelB TA
complexes, approximately a 30-residue C-terminal region of
YefM or RelB antitoxin, also mainly takes part in the binding
with YoeB or RelE toxin (18, 33), respectively, which are also
structural homologues of HP0892. Therefore, we hypothesized
that the C-terminal region of HP0893 is also responsible for
binding with HP0892 and investigated its binding with the
To confirm the binding of this C-terminal region of HP0893
PAGE of the elution fraction from the latter Ni2?-affinity column in the pull-
down assay (see “Experimental Procedures”) using a GST and a Ni2?-affinity
HP0892 and His-HP0893 co-expressed in cells.
FIGURE 3. Effects of expressing HP0892 and HP0893 on E. coli cell growth. A, viable counts of E. coli cells expressing the empty vector pET21a (?), the
expressions at the indicated induction times.
(Glu-66 to Leu-95), corresponding to the C-terminal region of
HP0893, and investigated the interaction between this peptide
and HP0892 with the NMR titration method. A series of two-
dimensional1H,15N TROSY spectra of15N-labeled HP0892
were recorded with successive additions of unlabeled
6, obvious chemical shift changes in slow or intermediate
exchange mode on the NMR time scale were observed for a lot
stants (Kd) of HP0893Ctp (and consequently HP0893) with
strong. Chemical shift changes from the residues in slow
exchange mode were completed with an equimolar amount of
HP0893Ctp. These results and the relative intensities of the
protein bands corresponding to HP0892 and HP0893 in the
cate that the binding stoichiometry of HP0892 and HP0893 is
1:1. Considering the overall results, we can reasonably suggest
ing with HP0892.
A plot of the chemical shift changes against the residues of
HP0892 is shown in Fig. 6B. For this plot, the backbone N and
various combinations of HP0892, HP0892 mutant (H86A), and/or HP0893.
4 ?M HP0893, respectively; 7, mRNA and 4 ?M HP0893.
FIGURE 5. Primer extension analyses of mRNA cleavage by HP0892. hp0893 mRNA with different DNA primers was used to produce A and B. Lanes 1–4,
mRNA. Lane 5, 2.5, 1.25, or 0 ?M, respectively, HP0892. DNA sequence ladders are shown at the left with upper lane labels for the sense strand. Major cleavage
RNA complement of the DNA sequencing ladder in the above images. The numbers and inverse triangles on the sequences correspond to the sequentially
numbered major cleavage sites in the above images.
HN resonances of uncomplexed HP0892 were assigned from
the data obtained in an earlier study (29). Regarding backbone
resonance assignments for HP0893Ctp-bound HP0892,
sequential C? connections determined from three-dimen-
sional trHNCA of13C,15N-labeled HP0892 in complex with
HP0893Ctp and comparison of C? resonance values between
FIGURE 6. Chemical shift perturbations of HP0892 residues by HP0893Ctp binding. A, two-dimensional1H,15N TROSY spectra of 100 ?M15N-labeled
HP0892 with HP0893Ctp added at 0 (black) and 1 (red) molar ratios of HP0893Ctp/HP0892 are superimposed. A central spectral region is enlarged aside. The
residues whose resonances disappeared (intermediate exchange mode) in the spectrum at 1 molar ratio are labeled in green. B, averaged chemical shift
changes (??ave) of individual residues of HP0892 upon binding with HP0893Ctp (1:1 molar ratio). The changes of the residues in the obvious slow exchange
mode (with ??avemore than 1.0 ppm) are colored in dark blue, those in the intermediate exchange mode in light red, and those with ??ave?1.0 ppm in light
the obvious slow or intermediate exchange mode are colored in red. The program PyMOL (35) was used to visualize the structures.
FEBRUARY22,2013•VOLUME288•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY 6009
uncomplexed and HP0893Ctp-complexed HP0892 were used.
The N-terminal residues Thr-3 to Asn-19, Asp-23, Lys-25,
Leu-27 to Val-30, Gly-32, Leu-34, Thr-35, and Gln-38, and the
56, and the C-terminal residues Leu-66, Ile-71, Lys-73, Glu-75,
Thr-77, Phe-79 to Leu-83, Ser-85, Ser-87, and Phe-88 showed
obvious chemical shift changes in the slow or intermediate
exchange mode. These three residue groups in the HP0892
sequence could be mapped structurally into a nearly consecu-
tive region on a three-dimensional HP0892 NMR structure
(Fig. 6C). It is reasonably suggested that those residue groups
form the binding site for HP0893. Overall, the chemical shift
changes of the residues in the N-terminal secondary structure
elements (one ?-strand and two ?-helixes in Leu-2 to Gln-38)
which indicates that this N-terminal region of HP0892 is the
main region responsible for its binding with HP0893.
Oligomerization State of HP0892-HP0893 Complex—To
HP0892-His and His-HP0893 was in the mixture) using 20 mM
mobile phase. As shown in Fig. 7, the HP0892-HP0893 (molar
73.69 kDa, which fit well with an oligomerization state of
(HP0892-HP0893)3heterohexamer (74.99 kDa).
In our previous study, based on the structural and sequential
similarities between H. pylori HP0892 and other RelE family
toxin molecules such as E. coli YoeB, Pyrococcus horikoshii
aRelE (the archaeal homologues of E. coli RelE), and H. pylori
HP0894, we reported that there is a reasonable possibility that
usually located directly upstream of the toxin gene, and on the
H. pylori chromosome, the hp0893 gene is located directly
upstream of the hp0892 gene (Fig. 1) in an operon. Therefore,
we expected that if HP0892 is a TA toxin, it would bind to
HP0893 from H. pylori 26695 shares no detectable sequence
similarity with other antitoxins. However, as revealed in the
mRNA, and HP0893 inhibits the decay of mRNA by HP0892.
Furthermore, His-86 of HP0892, which is homologous to key
catalytic residues of His-87 of YafQ (19), His-83 of YoeB (18),
(28), is also essential for its RNase activity. HP0892 expression
HP0893 neutralizes the cell toxicity of HP0892. Especially,
ity with YafQ toxin among the three E. coli RelE family toxins
yses, suggest that HP0892 is a YafQ-homologous toxin with
intrinsic RNase activity and HP0893 is the antitoxin against
The comparison of HP0892 with its structural homologues
harboring intrinsic RNase activity indicated that some of the
key catalytic (or putative catalytic) residues involved in the
RNase activity in RNase Sa, E. coli YoeB, and H. pylori HP0894
are conserved in HP0892 (Fig. 9). His-85 and Glu-54 of RNase
respectively. However, Arg-69 of RNase Sa (42) and Arg-65 of
YoeB (18), which form catalytic triads with the above two resi-
dues, respectively, are not conserved in HP0892 or HP0894
(28). Instead, Arg-82 of HP0892, like Arg-80 of HP0894 (28),
seems to play the role corresponding to those of Arg-69 of
RNase Sa or Arg-65 of YoeB.
By contrast, in the sequence homology analysis among
HP0892, HP0894, and YafQ, the residues forming putative cat-
alytic triad of HP0892 (Glu-58, Arg-82, and His-86) or HP0894
(Glu-58, Arg-80, and His-84) are relatively well conserved in
FIGURE 7. Gel filtration chromatogram of the HP0892-His/His-HP0893 mixture. Arrows above the chromatograms indicate the positions of the elution
corresponding to the HP0892-HP0893 complex is shown beside the elution peak.
YafQ (Asp-61, Arg-83, and His-87). However, whereas the Arg
or HP0894 is replaced with a slightly different residue, Asp-61
by Armalyate et al. (39), Asp-61 of YafQ plays a moderate role
for its catalytic activity. On the contrary, Asp-67 and His-50
both or either of the two residues was proposed to play the role
residues in YafQ compared with HP0892, HP0894, YoeB, and
RNase Sa might be due to the lack of absolute conservation of
(39), other catalytic residues of YafQ were also presented (Fig.
8). Overall, despite the small differences in local organization,
the amino acid residues forming active site (catalytic residues
and residues around them, some of which can take part in the
recognition of specific RNA sequence (29)) of YafQ are well
high probability that HP0892 and YafQ share nearly the same
functional mechanism. Moreover, HP0892, HP0894, and YafQ
commonly display a preference for purine immediately down-
stream from the cleavage site when cleaving mRNAs in vitro
organizations among them. These similarities in the active site
organization and the sequence specificity for RNA cleavage
confirm that HP0892 is a type of YafQ-homologous toxin, sim-
ilar to HP0894.
Our results show that the direct interaction of the HP0892-
HP0893 TA pair occurs mainly between the 30-residue C-ter-
minal tail of HP0893 and the N-terminal secondary structure
elements and an adjacent C-terminal region of HP0892. In
addition, some residues in the core region of HP0892 also take
pylori HP0894-HP0895, E. coli RelE-RelB, and YoeB-YefM
complexes (18, 28, 33). Overall, anchoring of the C-terminal
the toxin (33) is the common critical factor for strong binding
between the toxin and the antitoxin in RelE family TA com-
HP0895, and HP0892-HP0893 complexes.
However, there is a difference in the oligomeric state
between HP0892-HP0893 and other TA complex. In other TA
(44), or MazF-MazE (45) TA pairs in E. coli, the antitoxin mol-
H.pyloriHP0892,H.pyloriHP0894,E. coliYafQ,E. coliYoeB,andE. coliRelE.SequencealignmentwasdonewithClustalW(37)andcoloredaccordingtoBlosum
the functionally important catalytic residues of YafQ revealed by a mutagenesis analysis (39).
those of its structural homologues. Ribbon displays of H. pylori HP0892
(PDB ID 2OTR) (29) (A); H. pylori HP0894 (PDB ID 1Z8M) (40) (B); E. coli YoeB
(41, 42) (D) are shown. The labeled functional or predicted key residues (see
“Discussion”) are colored red. The structures were visualized by the program
HP0893 antitoxin exists in trimeric form, and the overall com-
Therefore, the HP0892-HP0893 TA complex can bind to their
promoter region on the chromosomal DNA with a different
mode than other TA complexes in the process of the negative
autoregulation of the transcription of their genes.
According to a study by Graham et al. (46), in common with
(28), hp0893 is also one of the H. pylori genomic open reading
frames that correspond to genes that are potentially expressed
in response to interactions with the human gastric mucosa. In
addition, according to gene comparison studies by Terry et al.
(47), both hp0892 and hp0893 belong to H. pylori genes absent
or novel virulence factors. Interestingly, until now, there have
been no other TA pairs in H. pylori 26695 except HP0892-
HP0893 and HP0894-HP0895 TA pairs, which have been pre-
(48). These indicate that the TA systems in H. pylori, especially
the HP0892-HP0893 pair, are related to the status of infections
of H. pylori in the human gastric mucosa, probably through
negative regulation of the toxin molecules by the antitoxins.
as the HP0894-HP0895 TA pair may be an appropriate new
target for antibacterial agents for H. pylori. The information in
this study on the binding aspect of these two proteins may be
helpful in the design and development of new antibiotic drugs.
Acknowledgments—We thank the National Center for Inter-Univer-
sity Research Facilities at Seoul National University and Korea Insti-
tute of Science and Technology for providing high field NMR
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