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Structural and biochemical characterization of HP0315 from Helicobacter pylori as a VapD protein with an endoribonuclease activity.

Kwon AR, Kim JH, Park SJ, Lee KY, Min YH, Im H, Lee I, Lee KY, Lee BJ - Nucleic Acids Res. (2012)

Bottom Line: VapD-like virulence-associated proteins have been found in many organisms, but little is known about this protein family including the 3D structure of these proteins.HP0315 is arranged as an operon with HP0316, which was found to be an antitoxin-related protein.Thus, HP0315 may be an evolutionary intermediate which does not belong to either the Cas2 family or TA system.

View Article: PubMed Central - PubMed

Affiliation: Department of Herbal Skin Care, College of Herbal Bio-Industry, Daegu Haany University, Gyeongsan 712-715, Korea.

ABSTRACT
VapD-like virulence-associated proteins have been found in many organisms, but little is known about this protein family including the 3D structure of these proteins. Recently, a relationship between the Cas2 family of ribonucleases associated with the CRISPR system of microbial immunity and VapD was suggested. Here, we show for the first time the structure of a member of the VapD family and present a relationship of VapD with Cas2 family and toxin-antitoxin (TA) systems. The crystal structure of HP0315 from Helicobacter pylori was solved at a resolution of 2.8 Å. The structure of HP0315, which has a modified ferredoxin-like fold, is very similar to that of the Cas2 family. Like Cas2 proteins, HP0315 shows endoribonuclease activity. HP0315-cleaved mRNA, mainly before A and G nucleotides preferentially, which means that HP0315 has purine-specific endoribonuclease activity. Mutagenesis studies of HP0315 revealed that D7, L13, S43 and D76 residues are important for RNase activity, in contrast, to the Cas2 family. HP0315 is arranged as an operon with HP0316, which was found to be an antitoxin-related protein. However, HP0315 is not a component of the TA system. Thus, HP0315 may be an evolutionary intermediate which does not belong to either the Cas2 family or TA system.

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Structure-based sequence analysis of HP0315 with other proteins belonging to VapD family and Cas2 proteins. (A) Residues conserved in all VapD family are colored under dark blue, highly conserved residues under medium blue and somewhat conserved residues under light blue. The secondary structure elements derived from the structure of HP0315 are displayed above the alignment. Residues marked with arrow were highly conserved among VapD family and selected as candidates for mutational studies. Residues with red arrow are related to catalytic activity based on mutational study. The compared proteins are as follows: HP0315 (NCBI accession ID: NP_207113), DNO_0265 (YP_001209119) from D. nodosus, HIAG_00714 (ZP_05850077) from H. influenza, NGK_p0009 (YP_002000622) from N. gonorrhoeae, PRU_1589 (YP_003574888) from P. ruminicola, VEIS_4994 (YP_999698) from V. diseniae, CAG_1570 (YP_379868) from C. chlorochromatii, HSM_1448 (YP_001784768) from H. somnus. (B) Structure-based sequence analysis of Cas2 proteins. Residues are colored like above sequence alignment. Residues marked with red box of SSO1404 are important for the catalytic activity of SSO1404 (26). The compared proteins are as follows: SSO1404 from S. solfataricus, SSO8090, TT1823 from T. thermophilus and PF1117 from P. furiosus. (C) Important residues for its activity of HP0315. According to mutational study, D7, L13, S43 and D76 are important residues for its activity. D7 and D76 are supposed to be a nucleophile which can attack and cleave phosphodiester bond of ssRNAs. L13 located on the α1 helix region can affect the local environment of α1 helix which is a candidate for first RNA-binding region using positive residues. S43 located between β2 and β3 might bind with uracil base, which might help the specific cleavage of RNA. (D) Important residues for its activity of SSO1404. D10 is known as a catalytic active site (26). (E) Superposition of HP0315 and SSO1404. HP0315 is colored by cyan and SSO1404 is colored by magenta. (F) Superposition of key residues of SSO1404 with the corresponding residues of HP0315. The sequences were aligned by CLUSTALW2 (53), and the figure was generated using Jalview (54).
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gkr1305-F3: Structure-based sequence analysis of HP0315 with other proteins belonging to VapD family and Cas2 proteins. (A) Residues conserved in all VapD family are colored under dark blue, highly conserved residues under medium blue and somewhat conserved residues under light blue. The secondary structure elements derived from the structure of HP0315 are displayed above the alignment. Residues marked with arrow were highly conserved among VapD family and selected as candidates for mutational studies. Residues with red arrow are related to catalytic activity based on mutational study. The compared proteins are as follows: HP0315 (NCBI accession ID: NP_207113), DNO_0265 (YP_001209119) from D. nodosus, HIAG_00714 (ZP_05850077) from H. influenza, NGK_p0009 (YP_002000622) from N. gonorrhoeae, PRU_1589 (YP_003574888) from P. ruminicola, VEIS_4994 (YP_999698) from V. diseniae, CAG_1570 (YP_379868) from C. chlorochromatii, HSM_1448 (YP_001784768) from H. somnus. (B) Structure-based sequence analysis of Cas2 proteins. Residues are colored like above sequence alignment. Residues marked with red box of SSO1404 are important for the catalytic activity of SSO1404 (26). The compared proteins are as follows: SSO1404 from S. solfataricus, SSO8090, TT1823 from T. thermophilus and PF1117 from P. furiosus. (C) Important residues for its activity of HP0315. According to mutational study, D7, L13, S43 and D76 are important residues for its activity. D7 and D76 are supposed to be a nucleophile which can attack and cleave phosphodiester bond of ssRNAs. L13 located on the α1 helix region can affect the local environment of α1 helix which is a candidate for first RNA-binding region using positive residues. S43 located between β2 and β3 might bind with uracil base, which might help the specific cleavage of RNA. (D) Important residues for its activity of SSO1404. D10 is known as a catalytic active site (26). (E) Superposition of HP0315 and SSO1404. HP0315 is colored by cyan and SSO1404 is colored by magenta. (F) Superposition of key residues of SSO1404 with the corresponding residues of HP0315. The sequences were aligned by CLUSTALW2 (53), and the figure was generated using Jalview (54).

Mentions: The molecular function of VapD is unknown. As a first step toward a functional evaluation, we performed a sequence-based homology search (Figure 3A). There are many sequence homologs among the proteins of the VapD family. In particular, residues in the first β-strand (β1), the loops between β2 and β3, and C-terminal region are highly conserved (Figure 3A). By considering that the C-terminal conserved region resulting from sequence alignments is related to dimerization, it is thought that the other region may contain active-site residues.Figure 3.


Structural and biochemical characterization of HP0315 from Helicobacter pylori as a VapD protein with an endoribonuclease activity.

Kwon AR, Kim JH, Park SJ, Lee KY, Min YH, Im H, Lee I, Lee KY, Lee BJ - Nucleic Acids Res. (2012)

Structure-based sequence analysis of HP0315 with other proteins belonging to VapD family and Cas2 proteins. (A) Residues conserved in all VapD family are colored under dark blue, highly conserved residues under medium blue and somewhat conserved residues under light blue. The secondary structure elements derived from the structure of HP0315 are displayed above the alignment. Residues marked with arrow were highly conserved among VapD family and selected as candidates for mutational studies. Residues with red arrow are related to catalytic activity based on mutational study. The compared proteins are as follows: HP0315 (NCBI accession ID: NP_207113), DNO_0265 (YP_001209119) from D. nodosus, HIAG_00714 (ZP_05850077) from H. influenza, NGK_p0009 (YP_002000622) from N. gonorrhoeae, PRU_1589 (YP_003574888) from P. ruminicola, VEIS_4994 (YP_999698) from V. diseniae, CAG_1570 (YP_379868) from C. chlorochromatii, HSM_1448 (YP_001784768) from H. somnus. (B) Structure-based sequence analysis of Cas2 proteins. Residues are colored like above sequence alignment. Residues marked with red box of SSO1404 are important for the catalytic activity of SSO1404 (26). The compared proteins are as follows: SSO1404 from S. solfataricus, SSO8090, TT1823 from T. thermophilus and PF1117 from P. furiosus. (C) Important residues for its activity of HP0315. According to mutational study, D7, L13, S43 and D76 are important residues for its activity. D7 and D76 are supposed to be a nucleophile which can attack and cleave phosphodiester bond of ssRNAs. L13 located on the α1 helix region can affect the local environment of α1 helix which is a candidate for first RNA-binding region using positive residues. S43 located between β2 and β3 might bind with uracil base, which might help the specific cleavage of RNA. (D) Important residues for its activity of SSO1404. D10 is known as a catalytic active site (26). (E) Superposition of HP0315 and SSO1404. HP0315 is colored by cyan and SSO1404 is colored by magenta. (F) Superposition of key residues of SSO1404 with the corresponding residues of HP0315. The sequences were aligned by CLUSTALW2 (53), and the figure was generated using Jalview (54).
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Related In: Results  -  Collection

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gkr1305-F3: Structure-based sequence analysis of HP0315 with other proteins belonging to VapD family and Cas2 proteins. (A) Residues conserved in all VapD family are colored under dark blue, highly conserved residues under medium blue and somewhat conserved residues under light blue. The secondary structure elements derived from the structure of HP0315 are displayed above the alignment. Residues marked with arrow were highly conserved among VapD family and selected as candidates for mutational studies. Residues with red arrow are related to catalytic activity based on mutational study. The compared proteins are as follows: HP0315 (NCBI accession ID: NP_207113), DNO_0265 (YP_001209119) from D. nodosus, HIAG_00714 (ZP_05850077) from H. influenza, NGK_p0009 (YP_002000622) from N. gonorrhoeae, PRU_1589 (YP_003574888) from P. ruminicola, VEIS_4994 (YP_999698) from V. diseniae, CAG_1570 (YP_379868) from C. chlorochromatii, HSM_1448 (YP_001784768) from H. somnus. (B) Structure-based sequence analysis of Cas2 proteins. Residues are colored like above sequence alignment. Residues marked with red box of SSO1404 are important for the catalytic activity of SSO1404 (26). The compared proteins are as follows: SSO1404 from S. solfataricus, SSO8090, TT1823 from T. thermophilus and PF1117 from P. furiosus. (C) Important residues for its activity of HP0315. According to mutational study, D7, L13, S43 and D76 are important residues for its activity. D7 and D76 are supposed to be a nucleophile which can attack and cleave phosphodiester bond of ssRNAs. L13 located on the α1 helix region can affect the local environment of α1 helix which is a candidate for first RNA-binding region using positive residues. S43 located between β2 and β3 might bind with uracil base, which might help the specific cleavage of RNA. (D) Important residues for its activity of SSO1404. D10 is known as a catalytic active site (26). (E) Superposition of HP0315 and SSO1404. HP0315 is colored by cyan and SSO1404 is colored by magenta. (F) Superposition of key residues of SSO1404 with the corresponding residues of HP0315. The sequences were aligned by CLUSTALW2 (53), and the figure was generated using Jalview (54).
Mentions: The molecular function of VapD is unknown. As a first step toward a functional evaluation, we performed a sequence-based homology search (Figure 3A). There are many sequence homologs among the proteins of the VapD family. In particular, residues in the first β-strand (β1), the loops between β2 and β3, and C-terminal region are highly conserved (Figure 3A). By considering that the C-terminal conserved region resulting from sequence alignments is related to dimerization, it is thought that the other region may contain active-site residues.Figure 3.

Bottom Line: VapD-like virulence-associated proteins have been found in many organisms, but little is known about this protein family including the 3D structure of these proteins.HP0315 is arranged as an operon with HP0316, which was found to be an antitoxin-related protein.Thus, HP0315 may be an evolutionary intermediate which does not belong to either the Cas2 family or TA system.

View Article: PubMed Central - PubMed

Affiliation: Department of Herbal Skin Care, College of Herbal Bio-Industry, Daegu Haany University, Gyeongsan 712-715, Korea.

ABSTRACT
VapD-like virulence-associated proteins have been found in many organisms, but little is known about this protein family including the 3D structure of these proteins. Recently, a relationship between the Cas2 family of ribonucleases associated with the CRISPR system of microbial immunity and VapD was suggested. Here, we show for the first time the structure of a member of the VapD family and present a relationship of VapD with Cas2 family and toxin-antitoxin (TA) systems. The crystal structure of HP0315 from Helicobacter pylori was solved at a resolution of 2.8 Å. The structure of HP0315, which has a modified ferredoxin-like fold, is very similar to that of the Cas2 family. Like Cas2 proteins, HP0315 shows endoribonuclease activity. HP0315-cleaved mRNA, mainly before A and G nucleotides preferentially, which means that HP0315 has purine-specific endoribonuclease activity. Mutagenesis studies of HP0315 revealed that D7, L13, S43 and D76 residues are important for RNase activity, in contrast, to the Cas2 family. HP0315 is arranged as an operon with HP0316, which was found to be an antitoxin-related protein. However, HP0315 is not a component of the TA system. Thus, HP0315 may be an evolutionary intermediate which does not belong to either the Cas2 family or TA system.

Show MeSH
Related in: MedlinePlus