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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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Cleavage experiments of small RNAs. Lanes M are imidazole ladder marker, which was prepared by partial RNA cleavage by imidazole. Lane 1 is 14 nt ssRNA without GB1_HP0315, and Lane 2 is 14 nt ssRNA with GB1_HP0315. Lane 3 is 12 nt ssRNA alone and Lane 4 is 12 nt ssRNA with GB1_HP0315. Lane 5 is 10 nt ssRNA alone and Lane 6 is 10 nt ssRNA with GB1_HP0315. Lane 7 is 8 nt ssRNA alone and Lane 8 is 8 nt ssRNA with GB1_HP0315. Lane 9 is 6 nt ssRNA alone and Lane 10 is 6 nt ssRNA with GB1_HP0315. Cleavage sites are indicated by closed triangles.
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gkr1305-F6: Cleavage experiments of small RNAs. Lanes M are imidazole ladder marker, which was prepared by partial RNA cleavage by imidazole. Lane 1 is 14 nt ssRNA without GB1_HP0315, and Lane 2 is 14 nt ssRNA with GB1_HP0315. Lane 3 is 12 nt ssRNA alone and Lane 4 is 12 nt ssRNA with GB1_HP0315. Lane 5 is 10 nt ssRNA alone and Lane 6 is 10 nt ssRNA with GB1_HP0315. Lane 7 is 8 nt ssRNA alone and Lane 8 is 8 nt ssRNA with GB1_HP0315. Lane 9 is 6 nt ssRNA alone and Lane 10 is 6 nt ssRNA with GB1_HP0315. Cleavage sites are indicated by closed triangles.

Mentions: To identify the limitation of the mRNA length which can be cleaved by GB1_HP0315, various short-length RNA templates (from 14 to 6 nt) were used for an RNase activity test (Figure 6). Initially, we tested the sequence (5′-GACUUUAGCGAUUU-3′: 14 nt) including a cleavable site, based on the primer extension results. Other small RNA templates were prepared by cutting 2 nt (1 nt at each terminal site). As shown in Figure 6, GB1_HP0315 could cleave at least 6 nt RNA. RNAs shorter than 6 nt were not tested due to the technical difficulty associated with this type of synthesis.Figure 6.


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)

Cleavage experiments of small RNAs. Lanes M are imidazole ladder marker, which was prepared by partial RNA cleavage by imidazole. Lane 1 is 14 nt ssRNA without GB1_HP0315, and Lane 2 is 14 nt ssRNA with GB1_HP0315. Lane 3 is 12 nt ssRNA alone and Lane 4 is 12 nt ssRNA with GB1_HP0315. Lane 5 is 10 nt ssRNA alone and Lane 6 is 10 nt ssRNA with GB1_HP0315. Lane 7 is 8 nt ssRNA alone and Lane 8 is 8 nt ssRNA with GB1_HP0315. Lane 9 is 6 nt ssRNA alone and Lane 10 is 6 nt ssRNA with GB1_HP0315. Cleavage sites are indicated by closed triangles.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351183&req=5

gkr1305-F6: Cleavage experiments of small RNAs. Lanes M are imidazole ladder marker, which was prepared by partial RNA cleavage by imidazole. Lane 1 is 14 nt ssRNA without GB1_HP0315, and Lane 2 is 14 nt ssRNA with GB1_HP0315. Lane 3 is 12 nt ssRNA alone and Lane 4 is 12 nt ssRNA with GB1_HP0315. Lane 5 is 10 nt ssRNA alone and Lane 6 is 10 nt ssRNA with GB1_HP0315. Lane 7 is 8 nt ssRNA alone and Lane 8 is 8 nt ssRNA with GB1_HP0315. Lane 9 is 6 nt ssRNA alone and Lane 10 is 6 nt ssRNA with GB1_HP0315. Cleavage sites are indicated by closed triangles.
Mentions: To identify the limitation of the mRNA length which can be cleaved by GB1_HP0315, various short-length RNA templates (from 14 to 6 nt) were used for an RNase activity test (Figure 6). Initially, we tested the sequence (5′-GACUUUAGCGAUUU-3′: 14 nt) including a cleavable site, based on the primer extension results. Other small RNA templates were prepared by cutting 2 nt (1 nt at each terminal site). As shown in Figure 6, GB1_HP0315 could cleave at least 6 nt RNA. RNAs shorter than 6 nt were not tested due to the technical difficulty associated with this type of synthesis.Figure 6.

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