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Crystal structure confirmation of JHP933 as a nucleotidyltransferase superfamily protein from Helicobacter pylori strain J99.

Zhao Y, Ye X, Su Y, Sun L, She F, Wu Y - PLoS ONE (2014)

Bottom Line: Studies suggested that certain genes in this region may play key roles in the pathogenesis of H. pylori-associated gastroduodenal diseases.A superposition demonstrates overall structural homology of the JHP933 N-terminal fragment with lincosamide antibiotic adenylyltransferase LinA and identifies a possible substrate-binding cleft of JHP933.Furthermore, through structural comparison with LinA and LinB, we pinpoint conservative active site residues which may contribute to divalent ion coordination and substrate binding.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian, China.

ABSTRACT
Helicobacter pylori is a well-known pathogen involved in the development of peptic ulcer, gastric adenocarcinoma and other forms of gastric cancer. Recently, there has been more considerable interest in strain-specific genes located in plasticity regions with great genetic variability. However, little is known about many of these genes. Studies suggested that certain genes in this region may play key roles in the pathogenesis of H. pylori-associated gastroduodenal diseases. JHP933, a conserved putative protein of unknown function, is encoded by the gene in plasticity region of H. pylori strain J99. Here we have determined the structure of JHP933. Our work demonstrates that JHP933 is a nucleotidyltransferase superfamily protein with a characteristic αβαβαβα topology. A superposition demonstrates overall structural homology of the JHP933 N-terminal fragment with lincosamide antibiotic adenylyltransferase LinA and identifies a possible substrate-binding cleft of JHP933. Furthermore, through structural comparison with LinA and LinB, we pinpoint conservative active site residues which may contribute to divalent ion coordination and substrate binding.

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Active site conservation and substrate binding of JHP933, LinA and LinB.The C atoms of active site residues are shown in ball-and-stick representation and distinctively colored: lime for JHP933, magenta for LinA (4E8J), and cyan for LinB (3JZ0). The substrate Mg2+ ions, as cyan spheres, AMPCPP and clindamycin, in yellow, are from LinB complex structure.
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pone-0104609-g005: Active site conservation and substrate binding of JHP933, LinA and LinB.The C atoms of active site residues are shown in ball-and-stick representation and distinctively colored: lime for JHP933, magenta for LinA (4E8J), and cyan for LinB (3JZ0). The substrate Mg2+ ions, as cyan spheres, AMPCPP and clindamycin, in yellow, are from LinB complex structure.

Mentions: Through sequence analyses of distinct members of NTase superfamily, a common sequence motif of active site residues has been noted: h[G/S], [D/E]h[D/E]h and h[D/E]h (h indicates a hydrophobic amino acid) [17]. The corresponding residues are G39, D55hD57 and E113 in JHP933; with G39 at the connection of β1 and α2, D55 and D57 located on β2, and E113 is placed on β5 structurally adjacent to β2 [Fig. 5]. To further clarify the active site and molecular mechanism for JHP933 substrate binding, we compared the structure of JHP933 N-terminal fragment with the LinA/lincomycin complex, in addition to another NTase fold protein LinB complexed with Mg2+, AMPCPP and clindamycin (pdb code: 3JZ0) [Fig. S3] [25]. The structural superpositions reveal that not only is the fold conserved but also position of catalytic residues. According to the superimposed structure of JHP933, the sites of G39, D55/D57 and E113 are strictly conserved 3-dimensionally [Fig. 5]. Conservation of the catalytic residues likely indicates a similar mechanism of action. Therefore, with reference to the structural conservation of these NTase superfamily proteins, the conserved G39 should play a crucial role in binding of substrates, and D55/D57 and E113 likely are involved in the coordination of divalent ions such as Mg2+, which chelates the phosphates of a nucleoside triphosphate substrate and plays a crucial role in activation of the second substrate's hydroxyl group [16], [25]. However, residues responsible for second substrate binding of LinB or LinA are not conserved in JHP933, likely reflecting differences in identity and structure of second substrates. However, the overall structure clearly confirms that JHP933 belongs to the NTase superfamily with the characteristic structural features well maintained.


Crystal structure confirmation of JHP933 as a nucleotidyltransferase superfamily protein from Helicobacter pylori strain J99.

Zhao Y, Ye X, Su Y, Sun L, She F, Wu Y - PLoS ONE (2014)

Active site conservation and substrate binding of JHP933, LinA and LinB.The C atoms of active site residues are shown in ball-and-stick representation and distinctively colored: lime for JHP933, magenta for LinA (4E8J), and cyan for LinB (3JZ0). The substrate Mg2+ ions, as cyan spheres, AMPCPP and clindamycin, in yellow, are from LinB complex structure.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125220&req=5

pone-0104609-g005: Active site conservation and substrate binding of JHP933, LinA and LinB.The C atoms of active site residues are shown in ball-and-stick representation and distinctively colored: lime for JHP933, magenta for LinA (4E8J), and cyan for LinB (3JZ0). The substrate Mg2+ ions, as cyan spheres, AMPCPP and clindamycin, in yellow, are from LinB complex structure.
Mentions: Through sequence analyses of distinct members of NTase superfamily, a common sequence motif of active site residues has been noted: h[G/S], [D/E]h[D/E]h and h[D/E]h (h indicates a hydrophobic amino acid) [17]. The corresponding residues are G39, D55hD57 and E113 in JHP933; with G39 at the connection of β1 and α2, D55 and D57 located on β2, and E113 is placed on β5 structurally adjacent to β2 [Fig. 5]. To further clarify the active site and molecular mechanism for JHP933 substrate binding, we compared the structure of JHP933 N-terminal fragment with the LinA/lincomycin complex, in addition to another NTase fold protein LinB complexed with Mg2+, AMPCPP and clindamycin (pdb code: 3JZ0) [Fig. S3] [25]. The structural superpositions reveal that not only is the fold conserved but also position of catalytic residues. According to the superimposed structure of JHP933, the sites of G39, D55/D57 and E113 are strictly conserved 3-dimensionally [Fig. 5]. Conservation of the catalytic residues likely indicates a similar mechanism of action. Therefore, with reference to the structural conservation of these NTase superfamily proteins, the conserved G39 should play a crucial role in binding of substrates, and D55/D57 and E113 likely are involved in the coordination of divalent ions such as Mg2+, which chelates the phosphates of a nucleoside triphosphate substrate and plays a crucial role in activation of the second substrate's hydroxyl group [16], [25]. However, residues responsible for second substrate binding of LinB or LinA are not conserved in JHP933, likely reflecting differences in identity and structure of second substrates. However, the overall structure clearly confirms that JHP933 belongs to the NTase superfamily with the characteristic structural features well maintained.

Bottom Line: Studies suggested that certain genes in this region may play key roles in the pathogenesis of H. pylori-associated gastroduodenal diseases.A superposition demonstrates overall structural homology of the JHP933 N-terminal fragment with lincosamide antibiotic adenylyltransferase LinA and identifies a possible substrate-binding cleft of JHP933.Furthermore, through structural comparison with LinA and LinB, we pinpoint conservative active site residues which may contribute to divalent ion coordination and substrate binding.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian, China.

ABSTRACT
Helicobacter pylori is a well-known pathogen involved in the development of peptic ulcer, gastric adenocarcinoma and other forms of gastric cancer. Recently, there has been more considerable interest in strain-specific genes located in plasticity regions with great genetic variability. However, little is known about many of these genes. Studies suggested that certain genes in this region may play key roles in the pathogenesis of H. pylori-associated gastroduodenal diseases. JHP933, a conserved putative protein of unknown function, is encoded by the gene in plasticity region of H. pylori strain J99. Here we have determined the structure of JHP933. Our work demonstrates that JHP933 is a nucleotidyltransferase superfamily protein with a characteristic αβαβαβα topology. A superposition demonstrates overall structural homology of the JHP933 N-terminal fragment with lincosamide antibiotic adenylyltransferase LinA and identifies a possible substrate-binding cleft of JHP933. Furthermore, through structural comparison with LinA and LinB, we pinpoint conservative active site residues which may contribute to divalent ion coordination and substrate binding.

Show MeSH
Related in: MedlinePlus