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 HP0892 may be a TA toxin. In TA systems, the antitoxin gene is 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 () in an operon. Therefore, we expected that if HP0892 is a TA toxin, it would bind to HP0893. The 95-residue H. pylori
-specific hypothetical protein HP0893 from H. pylori
26695 shares no detectable sequence similarity with other antitoxins. However, as revealed in the present study, HP0892 strongly binds to HP0893, with presumably a 1:1 binding stoichiometry. HP0892 has RNase activity on 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
), Arg-81 of RelE (33
), Arg-85 of aRelE (36
), and His-84 of HP0894 (28
), is also essential for its RNase activity. HP0892 expression has a toxic effect on E. coli
cell growth, but the co-expression of HP0893 neutralizes the cell toxicity of HP0892. Especially, HP0892 shares detectable and relatively high sequence similarity with YafQ toxin among the three E. coli
RelE family toxins (). These results, including sequential and structural analyses, suggest that HP0892 is a YafQ-homologous toxin with intrinsic RNase activity and HP0893 is the antitoxin against HP0892.
FIGURE 8. Sequence homology of HP0892.
A, multiple sequence alignments of H. pylori HP0892, H. pylori HP0894, and E. coli YafQ. B, average distance tree of H. pylori HP0892, H. pylori HP0894, E. coli YafQ, E. coli YoeB, and E. coli RelE. Sequence alignment was (more ...)
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 (). His-85 and Glu-54 of RNase Sa (42
), His-83 and Glu-46 of YoeB (18
), and His-84 and Glu-58 in HP0894 (28
) are replaced with His-86 and Glu-58 in HP0892, respectively. However, Arg-69 of RNase Sa (42
) and Arg-65 of YoeB (18
), which form catalytic triads with the above two residues, 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.
FIGURE 9. Comparison of structural and catalytic residues of HP0892 with 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 (PDB ID 2A6R) (18) (C); and Streptomyces (more ...)
By contrast, in the sequence homology analysis among HP0892, HP0894, and YafQ, the residues forming putative catalytic triad of HP0892 (Glu-58, Arg-82, and His-86) or HP0894 (Glu-58, Arg-80, and His-84) are relatively well conserved in YafQ (Asp-61, Arg-83, and His-87). However, whereas the Arg and His residues are absolutely conserved among them, the Glu residue, which seems to play the role of general base, of HP0892 or HP0894 is replaced with a slightly different residue, Asp-61 in YafQ. According to a mutagenesis study on E. coli
YafQ toxin by Armalyate et al.
), Asp-61 of YafQ plays a moderate role for its catalytic activity. On the contrary, Asp-67 and His-50 were shown to be critical for the catalytic activity of YafQ; thus, both or either of the two residues was proposed to play the role of general base instead of Asp-61. The above change of catalytic residues in YafQ compared with HP0892, HP0894, YoeB, and RNase Sa might be due to the lack of absolute conservation of Glu as the general base residue. In the study by Armalyate et al.
), other catalytic residues of YafQ were also presented (). 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 conserved in HP0892 and HP0894 (). Therefore, there is a high probability that HP0892 and YafQ share nearly the same functional mechanism. Moreover, HP0892, HP0894, and YafQ commonly display a preference for purine immediately downstream from the cleavage site when cleaving mRNAs in vitro
), which can explain the result of the similar active site 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, similar to HP0894.
Our results show that the direct interaction of the HP0892-HP0893 TA pair occurs mainly between the 30-residue C-terminal 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 part in the interaction. Similar interaction modes are seen in H. pylori
HP0894-HP0895, E. coli
RelE-RelB, and YoeB-YefM complexes (18
). Overall, anchoring of the C-terminal region of the antitoxin to the N-terminal secondary structure of the toxin (33
) is the common critical factor for strong binding between the toxin and the antitoxin in RelE family TA complexes such as E. coli
RelE-RelB, YoeB-YefM, H. pylori
HP0894-HP0895, and HP0892-HP0893 complexes.
However, there is a difference in the oligomeric state between HP0892-HP0893 and other TA complex. In other TA complexes such as RelE-RelB (43
), YoeB-YefM (18
), YafQ-DinJ (44
), or MazF-MazE (45
) TA pairs in E. coli
, the antitoxin molecules exist in dimeric form. The dimeric form of the antitoxins is related to the DNA binding mode of those TA complexes (36
). By contrast, in the HP0892-HP0893 complex, it seems that HP0893 antitoxin exists in trimeric form, and the overall complex has a heterohexameric arrangement ((HP0892-HP0893)3
). 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.
), in common with hp0895
(the antitoxin gene in the HP0894/HP0895 TA system) (28
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.
), both hp0892
belong to H. pylori
genes absent from a set of five-cag pathogenicity island-negative strains, and may represent a marker for the identification of virulent strains 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 predicted from bioinformatics studies or identified experimentally (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.
Because TA systems exist only in prokaryote and are directly involved in cell death, TA systems are promising antibiotic drug targets. Thus, the HP0892-HP0893 TA pair in this study as well 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.