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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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Enzymatic activity experiments. (A) Lane 1 is ssRNA alone. Lane 2 is ssRNA with GB1_HP0315. Lane 3 is dsDNA with GB1_HP0315. Lane 4 is ssDNA with GB1_HP0315. (B) Lane C is RNA alone, lane 1 is at pH 7.0, lane 2 at pH 7.5, lane 3 at pH 8.0, lane 4 at pH 8.5, lane 5 at pH 9.0, lane 6 at pH 9.5, lane 7 at pH 10.0 and lane 8 at pH 11.0. Some precipitations are occurred at and below pH 6.0. (C) Lane C is RNA alone and lane 1 is RNA and GB1_HP0315 without divalent cation. Lane 2 is RNA and GB1_HP0315 with 2.5 mM Mg2+ ion, lane 3 with 2.5 mM Mn2+, lane 4 with 2.5 mM Co2+ and lane 5 with 2.5 mM Ca2+ (D) Lane C is RNA alone. Lane 1 is RNA and GB1_HP0315 with no monovalent ion. Lane 2 is RNA and HP0315 with 100 mM NaCl, lane 3 is with 200 mM NaCl, lane 4 is with 300 mM NaCl, lane 5 is with 400 mM NaCl and lane 6 is with 500 mM NaCl. Lane 7 is RNA and HP0315 with 100 mM KCl, lane 8 is with 200 mM KCl, lane 9 is 300 mM KCl and lane 10 is with 400 mM KCl. (E) Ribonuclease activity of mutated GB1_HP0315 using fluorescence. A fluorescent substrate is incubated with various mutated GB1_HP0315 in same condition. When fluorescence-quenched oligonucleotide probes are cleaved, fluorescence can be appeared in the process of time. Wild-type protein of HP0315 cleaved the substrate most quickly. F6A, Y25A, Q41A, Y45A, W39A, D76A, D7A, S43A and L13A showed highest RNase activity in order. This result clearly indicates that D7, L13, S43 and D76 are most important residues for ribonuclease activity.
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gkr1305-F4: Enzymatic activity experiments. (A) Lane 1 is ssRNA alone. Lane 2 is ssRNA with GB1_HP0315. Lane 3 is dsDNA with GB1_HP0315. Lane 4 is ssDNA with GB1_HP0315. (B) Lane C is RNA alone, lane 1 is at pH 7.0, lane 2 at pH 7.5, lane 3 at pH 8.0, lane 4 at pH 8.5, lane 5 at pH 9.0, lane 6 at pH 9.5, lane 7 at pH 10.0 and lane 8 at pH 11.0. Some precipitations are occurred at and below pH 6.0. (C) Lane C is RNA alone and lane 1 is RNA and GB1_HP0315 without divalent cation. Lane 2 is RNA and GB1_HP0315 with 2.5 mM Mg2+ ion, lane 3 with 2.5 mM Mn2+, lane 4 with 2.5 mM Co2+ and lane 5 with 2.5 mM Ca2+ (D) Lane C is RNA alone. Lane 1 is RNA and GB1_HP0315 with no monovalent ion. Lane 2 is RNA and HP0315 with 100 mM NaCl, lane 3 is with 200 mM NaCl, lane 4 is with 300 mM NaCl, lane 5 is with 400 mM NaCl and lane 6 is with 500 mM NaCl. Lane 7 is RNA and HP0315 with 100 mM KCl, lane 8 is with 200 mM KCl, lane 9 is 300 mM KCl and lane 10 is with 400 mM KCl. (E) Ribonuclease activity of mutated GB1_HP0315 using fluorescence. A fluorescent substrate is incubated with various mutated GB1_HP0315 in same condition. When fluorescence-quenched oligonucleotide probes are cleaved, fluorescence can be appeared in the process of time. Wild-type protein of HP0315 cleaved the substrate most quickly. F6A, Y25A, Q41A, Y45A, W39A, D76A, D7A, S43A and L13A showed highest RNase activity in order. This result clearly indicates that D7, L13, S43 and D76 are most important residues for ribonuclease activity.

Mentions: The results presented in the previous section showed that HP0315 may display ribonuclease activity, similar to Cas2 proteins. To identify the biochemical activity of HP0315, we performed a primer extension experiment as well as agarose-based nuclease experiments. The purified protein and various nucleotide substrates including linear double-stranded DNA (dsDNA: PCR amplified of hp0315 gene, 308 nt), linear single-stranded DNA (ssDNA: C-terminal oligonucleotide of hp0315 gene, 42 nt) and single-stranded RNA (ssRNA: in vitro transcript of hp0315 gene, 308 nt) were used to test for nuclease activity (Figure 4A). Interestingly, GB1_HP0315 cleaved only ssRNA among them. In addition, GB1_HP0315 showed RNase activity over a broad range of pH with the maximum activity at pH 7.0–7.5 (Figure 4B). Although some nucleases, including Cas2 proteins, show the highest level of activity in the presence of divalent cations, e.g. Mg2+ and Mn2+ (26,43,44), the ribonuclease activity of GB1_HP0315 was not dependent on metal ions with ranges of Mg2+ and Mn2+ concentrations of 1, 10, 50, 100 μM, 1 and 2.5 mM (Figure 4C) (data are not shown). In addition to divalent ions, the RNase activity of GB1_HP0315 was not influenced by monovalent ions. We performed agarose-based electrophoretic mobility shift assays according to the concentration of the monovalent ions of Na+ or K+ ions from 0 to 400 mM. However, in the case of GB1_HP0315, the RNase activity was not significantly influenced by the Na+ or the K+ ions (Figure 4D).Figure 4.


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)

Enzymatic activity experiments. (A) Lane 1 is ssRNA alone. Lane 2 is ssRNA with GB1_HP0315. Lane 3 is dsDNA with GB1_HP0315. Lane 4 is ssDNA with GB1_HP0315. (B) Lane C is RNA alone, lane 1 is at pH 7.0, lane 2 at pH 7.5, lane 3 at pH 8.0, lane 4 at pH 8.5, lane 5 at pH 9.0, lane 6 at pH 9.5, lane 7 at pH 10.0 and lane 8 at pH 11.0. Some precipitations are occurred at and below pH 6.0. (C) Lane C is RNA alone and lane 1 is RNA and GB1_HP0315 without divalent cation. Lane 2 is RNA and GB1_HP0315 with 2.5 mM Mg2+ ion, lane 3 with 2.5 mM Mn2+, lane 4 with 2.5 mM Co2+ and lane 5 with 2.5 mM Ca2+ (D) Lane C is RNA alone. Lane 1 is RNA and GB1_HP0315 with no monovalent ion. Lane 2 is RNA and HP0315 with 100 mM NaCl, lane 3 is with 200 mM NaCl, lane 4 is with 300 mM NaCl, lane 5 is with 400 mM NaCl and lane 6 is with 500 mM NaCl. Lane 7 is RNA and HP0315 with 100 mM KCl, lane 8 is with 200 mM KCl, lane 9 is 300 mM KCl and lane 10 is with 400 mM KCl. (E) Ribonuclease activity of mutated GB1_HP0315 using fluorescence. A fluorescent substrate is incubated with various mutated GB1_HP0315 in same condition. When fluorescence-quenched oligonucleotide probes are cleaved, fluorescence can be appeared in the process of time. Wild-type protein of HP0315 cleaved the substrate most quickly. F6A, Y25A, Q41A, Y45A, W39A, D76A, D7A, S43A and L13A showed highest RNase activity in order. This result clearly indicates that D7, L13, S43 and D76 are most important residues for ribonuclease activity.
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gkr1305-F4: Enzymatic activity experiments. (A) Lane 1 is ssRNA alone. Lane 2 is ssRNA with GB1_HP0315. Lane 3 is dsDNA with GB1_HP0315. Lane 4 is ssDNA with GB1_HP0315. (B) Lane C is RNA alone, lane 1 is at pH 7.0, lane 2 at pH 7.5, lane 3 at pH 8.0, lane 4 at pH 8.5, lane 5 at pH 9.0, lane 6 at pH 9.5, lane 7 at pH 10.0 and lane 8 at pH 11.0. Some precipitations are occurred at and below pH 6.0. (C) Lane C is RNA alone and lane 1 is RNA and GB1_HP0315 without divalent cation. Lane 2 is RNA and GB1_HP0315 with 2.5 mM Mg2+ ion, lane 3 with 2.5 mM Mn2+, lane 4 with 2.5 mM Co2+ and lane 5 with 2.5 mM Ca2+ (D) Lane C is RNA alone. Lane 1 is RNA and GB1_HP0315 with no monovalent ion. Lane 2 is RNA and HP0315 with 100 mM NaCl, lane 3 is with 200 mM NaCl, lane 4 is with 300 mM NaCl, lane 5 is with 400 mM NaCl and lane 6 is with 500 mM NaCl. Lane 7 is RNA and HP0315 with 100 mM KCl, lane 8 is with 200 mM KCl, lane 9 is 300 mM KCl and lane 10 is with 400 mM KCl. (E) Ribonuclease activity of mutated GB1_HP0315 using fluorescence. A fluorescent substrate is incubated with various mutated GB1_HP0315 in same condition. When fluorescence-quenched oligonucleotide probes are cleaved, fluorescence can be appeared in the process of time. Wild-type protein of HP0315 cleaved the substrate most quickly. F6A, Y25A, Q41A, Y45A, W39A, D76A, D7A, S43A and L13A showed highest RNase activity in order. This result clearly indicates that D7, L13, S43 and D76 are most important residues for ribonuclease activity.
Mentions: The results presented in the previous section showed that HP0315 may display ribonuclease activity, similar to Cas2 proteins. To identify the biochemical activity of HP0315, we performed a primer extension experiment as well as agarose-based nuclease experiments. The purified protein and various nucleotide substrates including linear double-stranded DNA (dsDNA: PCR amplified of hp0315 gene, 308 nt), linear single-stranded DNA (ssDNA: C-terminal oligonucleotide of hp0315 gene, 42 nt) and single-stranded RNA (ssRNA: in vitro transcript of hp0315 gene, 308 nt) were used to test for nuclease activity (Figure 4A). Interestingly, GB1_HP0315 cleaved only ssRNA among them. In addition, GB1_HP0315 showed RNase activity over a broad range of pH with the maximum activity at pH 7.0–7.5 (Figure 4B). Although some nucleases, including Cas2 proteins, show the highest level of activity in the presence of divalent cations, e.g. Mg2+ and Mn2+ (26,43,44), the ribonuclease activity of GB1_HP0315 was not dependent on metal ions with ranges of Mg2+ and Mn2+ concentrations of 1, 10, 50, 100 μM, 1 and 2.5 mM (Figure 4C) (data are not shown). In addition to divalent ions, the RNase activity of GB1_HP0315 was not influenced by monovalent ions. We performed agarose-based electrophoretic mobility shift assays according to the concentration of the monovalent ions of Na+ or K+ ions from 0 to 400 mM. However, in the case of GB1_HP0315, the RNase activity was not significantly influenced by the Na+ or the K+ ions (Figure 4D).Figure 4.

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