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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 of HP0315 from H. pylori. (A) Cartoon representation of dimer of HP0315 (α-helices, β-strands and loops are cyan, magenta and yellow, respectively). Dotted circle represents the putative catalytic region located at deep cavity region. (B) Superposition of molecules A and B from HP0315 with an average r.m.s.d. of 0.362 Å. Two molecules are slightly different from each other in the loop between β2 and β3 and C-terminal region. (C) Surface representation of HP0315 showing positive electrostatic potential in blue and negative in red. Dotted circle represents the putative RNA-binding region. This region would be related to initial binding with RNA, and then second catalytic reaction would be happen around deep cavity region. (D) The image seeing down from top indicates that highly conserved residues (F6, L13, Y25, F37, Y45 and V74) from the result of structure based sequence alignment are concerned with hydrophobic interaction. Hydrophobic residues are colored in red and hydrophilic residues are in blue, respectively. (E) This figure shows that clustered hydrophobic residues into inside form the hydrophobic interaction core. All figures were prepared using PyMOL (52).
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gkr1305-F1: Structure of HP0315 from H. pylori. (A) Cartoon representation of dimer of HP0315 (α-helices, β-strands and loops are cyan, magenta and yellow, respectively). Dotted circle represents the putative catalytic region located at deep cavity region. (B) Superposition of molecules A and B from HP0315 with an average r.m.s.d. of 0.362 Å. Two molecules are slightly different from each other in the loop between β2 and β3 and C-terminal region. (C) Surface representation of HP0315 showing positive electrostatic potential in blue and negative in red. Dotted circle represents the putative RNA-binding region. This region would be related to initial binding with RNA, and then second catalytic reaction would be happen around deep cavity region. (D) The image seeing down from top indicates that highly conserved residues (F6, L13, Y25, F37, Y45 and V74) from the result of structure based sequence alignment are concerned with hydrophobic interaction. Hydrophobic residues are colored in red and hydrophilic residues are in blue, respectively. (E) This figure shows that clustered hydrophobic residues into inside form the hydrophobic interaction core. All figures were prepared using PyMOL (52).

Mentions: The dimeric structure of HP0315 fused with a GB1 protein at each N-terminus was determined at a resolution of 2.8 Å. The structure of HP0315 from H. pylori consists of 10 secondary structure elements: β1 (residues 1–8), α1 (residues 10–17), α1′ (residues 21–35), β2 (residues 38–41), β3 (residues 44–47), α2 (residues 53–66), α2′ (residues 68–73), β4 (residues 75–87) and α3 (residues 88–93). The monomer has a ferredoxin-like fold. It has the β1-(α1-α1′)-β2 -β3-(α2-α2′)-β4-α3 instead of the β-α-β-β-α-β structure of the ferredoxin fold. In the first half of the monomer, α1 is shorter than α1′ and, in the last half of the monomer, α2 is longer than α2′. The α1′ and α2 helices have amphipathic property, and the hydrophobic side of these amphipathic helices faces the inner space. Many hydrophobic residues facing the protein interior form a hydrophobic core for structural stability, which give the structure components stability (Figure 1D and E). In addition, the β strands are stabilized by direct hydrogen bonds between neighboring β-strands.Figure 1.


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 of HP0315 from H. pylori. (A) Cartoon representation of dimer of HP0315 (α-helices, β-strands and loops are cyan, magenta and yellow, respectively). Dotted circle represents the putative catalytic region located at deep cavity region. (B) Superposition of molecules A and B from HP0315 with an average r.m.s.d. of 0.362 Å. Two molecules are slightly different from each other in the loop between β2 and β3 and C-terminal region. (C) Surface representation of HP0315 showing positive electrostatic potential in blue and negative in red. Dotted circle represents the putative RNA-binding region. This region would be related to initial binding with RNA, and then second catalytic reaction would be happen around deep cavity region. (D) The image seeing down from top indicates that highly conserved residues (F6, L13, Y25, F37, Y45 and V74) from the result of structure based sequence alignment are concerned with hydrophobic interaction. Hydrophobic residues are colored in red and hydrophilic residues are in blue, respectively. (E) This figure shows that clustered hydrophobic residues into inside form the hydrophobic interaction core. All figures were prepared using PyMOL (52).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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gkr1305-F1: Structure of HP0315 from H. pylori. (A) Cartoon representation of dimer of HP0315 (α-helices, β-strands and loops are cyan, magenta and yellow, respectively). Dotted circle represents the putative catalytic region located at deep cavity region. (B) Superposition of molecules A and B from HP0315 with an average r.m.s.d. of 0.362 Å. Two molecules are slightly different from each other in the loop between β2 and β3 and C-terminal region. (C) Surface representation of HP0315 showing positive electrostatic potential in blue and negative in red. Dotted circle represents the putative RNA-binding region. This region would be related to initial binding with RNA, and then second catalytic reaction would be happen around deep cavity region. (D) The image seeing down from top indicates that highly conserved residues (F6, L13, Y25, F37, Y45 and V74) from the result of structure based sequence alignment are concerned with hydrophobic interaction. Hydrophobic residues are colored in red and hydrophilic residues are in blue, respectively. (E) This figure shows that clustered hydrophobic residues into inside form the hydrophobic interaction core. All figures were prepared using PyMOL (52).
Mentions: The dimeric structure of HP0315 fused with a GB1 protein at each N-terminus was determined at a resolution of 2.8 Å. The structure of HP0315 from H. pylori consists of 10 secondary structure elements: β1 (residues 1–8), α1 (residues 10–17), α1′ (residues 21–35), β2 (residues 38–41), β3 (residues 44–47), α2 (residues 53–66), α2′ (residues 68–73), β4 (residues 75–87) and α3 (residues 88–93). The monomer has a ferredoxin-like fold. It has the β1-(α1-α1′)-β2 -β3-(α2-α2′)-β4-α3 instead of the β-α-β-β-α-β structure of the ferredoxin fold. In the first half of the monomer, α1 is shorter than α1′ and, in the last half of the monomer, α2 is longer than α2′. The α1′ and α2 helices have amphipathic property, and the hydrophobic side of these amphipathic helices faces the inner space. Many hydrophobic residues facing the protein interior form a hydrophobic core for structural stability, which give the structure components stability (Figure 1D and E). In addition, the β strands are stabilized by direct hydrogen bonds between neighboring β-strands.Figure 1.

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