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Structural characterization of the virulence factor nuclease A from Streptococcus agalactiae.

Moon AF, Gaudu P, Pedersen LC - Acta Crystallogr. D Biol. Crystallogr. (2014)

Bottom Line: Several mutants on the surface of GBS_NucA which might influence DNA substrate binding and catalysis were generated and evaluated using an imidazole chemical rescue technique.While several of these mutants severely inhibited nuclease activity, two mutants (K146R and Q183A) exhibited significantly increased activity.These structural and biochemical studies have greatly increased our understanding of the mechanism of action of GBS_NucA in bacterial virulence and may serve as a foundation for the structure-based drug design of antibacterial compounds targeted to S. agalactiae.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.

ABSTRACT
The group B pathogen Streptococcus agalactiae commonly populates the human gut and urogenital tract, and is a major cause of infection-based mortality in neonatal infants and in elderly or immunocompromised adults. Nuclease A (GBS_NucA), a secreted DNA/RNA nuclease, serves as a virulence factor for S. agalactiae, facilitating bacterial evasion of the human innate immune response. GBS_NucA efficiently degrades the DNA matrix component of neutrophil extracellular traps (NETs), which attempt to kill and clear invading bacteria during the early stages of infection. In order to better understand the mechanisms of DNA substrate binding and catalysis of GBS_NucA, the high-resolution structure of a catalytically inactive mutant (H148G) was solved by X-ray crystallography. Several mutants on the surface of GBS_NucA which might influence DNA substrate binding and catalysis were generated and evaluated using an imidazole chemical rescue technique. While several of these mutants severely inhibited nuclease activity, two mutants (K146R and Q183A) exhibited significantly increased activity. These structural and biochemical studies have greatly increased our understanding of the mechanism of action of GBS_NucA in bacterial virulence and may serve as a foundation for the structure-based drug design of antibacterial compounds targeted to S. agalactiae.

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Identification of NucA surface residues for mutagenesis and biochemical characterization. (a) Least-squares superposition (LSQ) of the ββα motifs from GBS_NucA (H148A) (Val144–Gly147, Val171–Thr175 and Ala176–Asn179, blue) and VVN nuclease (PDB entry 2ivk; Wang et al., 2007 ▶; Ile76–Glu79, Leu119–Ile123 and Gly124–Asn127, brown). (b) Modeling of the 16 bp duplex DNA substrate (orange) from VVN nuclease onto the structure of GBS_NucA (H148A) (blue, molecular surface in gray) based on the LSQ superposition of their ββα motifs. The scissile DNA strand is shown in orange, with the location of the scissile phosphate highlighted in green. The complementary DNA strand is shown in yellow. (c) GBS_NucA (H148A) surface residues chosen for mutagenesis with likelihood of involvement in catalysis (red) or DNA substrate binding (cyan or purple).
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fig5: Identification of NucA surface residues for mutagenesis and biochemical characterization. (a) Least-squares superposition (LSQ) of the ββα motifs from GBS_NucA (H148A) (Val144–Gly147, Val171–Thr175 and Ala176–Asn179, blue) and VVN nuclease (PDB entry 2ivk; Wang et al., 2007 ▶; Ile76–Glu79, Leu119–Ile123 and Gly124–Asn127, brown). (b) Modeling of the 16 bp duplex DNA substrate (orange) from VVN nuclease onto the structure of GBS_NucA (H148A) (blue, molecular surface in gray) based on the LSQ superposition of their ββα motifs. The scissile DNA strand is shown in orange, with the location of the scissile phosphate highlighted in green. The complementary DNA strand is shown in yellow. (c) GBS_NucA (H148A) surface residues chosen for mutagenesis with likelihood of involvement in catalysis (red) or DNA substrate binding (cyan or purple).

Mentions: Thus far, there are no published structures of EndA, Spd1 or other similar ββα metal-finger nucleases in complex with a bound DNA or RNA substrate. Similar attempts to co-crystallize such a complex with GBS_NucA have likewise failed (unpublished data; see Supporting Information). However, X-ray crystal structures exist for the ββα-Me periplasmic nuclease VVN from Vibrio vulnificus in complex with duplex DNA [PDB entries 1oup (Li et al., 2003 ▶) and 2ivk (Wang et al., 2007 ▶)]. VVN is unreleated to the Streptococcus sp. ββα-Me nucleases and exhibits no sequence conservation. Alhough the DALI server was unable to identify any discernable structural homology with VVN, superposition of the ββα motif alone (Glu77–Ala80, Thr120–Ile123 and Gly124–Asn127 in VVN) yields admirable results (Fig. 5 ▶a). Using this alignment, the DNA substrates bound to VVN were docked onto the surface of NucA (Fig. 5 ▶b) and lie along a shallow cleft in close contact with the active site. The ‘front’ face of NucA was then surveyed for surface-accessible residues proximal to the active site which might have the capacity to influence catalysis (Fig. 5 ▶c, red) or to interact with the DNA substrate (Fig. 5 ▶c, cyan or purple). These residues were subjected to alanine-substitution mutagenesis and were subsequently assayed for nuclease activity.


Structural characterization of the virulence factor nuclease A from Streptococcus agalactiae.

Moon AF, Gaudu P, Pedersen LC - Acta Crystallogr. D Biol. Crystallogr. (2014)

Identification of NucA surface residues for mutagenesis and biochemical characterization. (a) Least-squares superposition (LSQ) of the ββα motifs from GBS_NucA (H148A) (Val144–Gly147, Val171–Thr175 and Ala176–Asn179, blue) and VVN nuclease (PDB entry 2ivk; Wang et al., 2007 ▶; Ile76–Glu79, Leu119–Ile123 and Gly124–Asn127, brown). (b) Modeling of the 16 bp duplex DNA substrate (orange) from VVN nuclease onto the structure of GBS_NucA (H148A) (blue, molecular surface in gray) based on the LSQ superposition of their ββα motifs. The scissile DNA strand is shown in orange, with the location of the scissile phosphate highlighted in green. The complementary DNA strand is shown in yellow. (c) GBS_NucA (H148A) surface residues chosen for mutagenesis with likelihood of involvement in catalysis (red) or DNA substrate binding (cyan or purple).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4220975&req=5

fig5: Identification of NucA surface residues for mutagenesis and biochemical characterization. (a) Least-squares superposition (LSQ) of the ββα motifs from GBS_NucA (H148A) (Val144–Gly147, Val171–Thr175 and Ala176–Asn179, blue) and VVN nuclease (PDB entry 2ivk; Wang et al., 2007 ▶; Ile76–Glu79, Leu119–Ile123 and Gly124–Asn127, brown). (b) Modeling of the 16 bp duplex DNA substrate (orange) from VVN nuclease onto the structure of GBS_NucA (H148A) (blue, molecular surface in gray) based on the LSQ superposition of their ββα motifs. The scissile DNA strand is shown in orange, with the location of the scissile phosphate highlighted in green. The complementary DNA strand is shown in yellow. (c) GBS_NucA (H148A) surface residues chosen for mutagenesis with likelihood of involvement in catalysis (red) or DNA substrate binding (cyan or purple).
Mentions: Thus far, there are no published structures of EndA, Spd1 or other similar ββα metal-finger nucleases in complex with a bound DNA or RNA substrate. Similar attempts to co-crystallize such a complex with GBS_NucA have likewise failed (unpublished data; see Supporting Information). However, X-ray crystal structures exist for the ββα-Me periplasmic nuclease VVN from Vibrio vulnificus in complex with duplex DNA [PDB entries 1oup (Li et al., 2003 ▶) and 2ivk (Wang et al., 2007 ▶)]. VVN is unreleated to the Streptococcus sp. ββα-Me nucleases and exhibits no sequence conservation. Alhough the DALI server was unable to identify any discernable structural homology with VVN, superposition of the ββα motif alone (Glu77–Ala80, Thr120–Ile123 and Gly124–Asn127 in VVN) yields admirable results (Fig. 5 ▶a). Using this alignment, the DNA substrates bound to VVN were docked onto the surface of NucA (Fig. 5 ▶b) and lie along a shallow cleft in close contact with the active site. The ‘front’ face of NucA was then surveyed for surface-accessible residues proximal to the active site which might have the capacity to influence catalysis (Fig. 5 ▶c, red) or to interact with the DNA substrate (Fig. 5 ▶c, cyan or purple). These residues were subjected to alanine-substitution mutagenesis and were subsequently assayed for nuclease activity.

Bottom Line: Several mutants on the surface of GBS_NucA which might influence DNA substrate binding and catalysis were generated and evaluated using an imidazole chemical rescue technique.While several of these mutants severely inhibited nuclease activity, two mutants (K146R and Q183A) exhibited significantly increased activity.These structural and biochemical studies have greatly increased our understanding of the mechanism of action of GBS_NucA in bacterial virulence and may serve as a foundation for the structure-based drug design of antibacterial compounds targeted to S. agalactiae.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.

ABSTRACT
The group B pathogen Streptococcus agalactiae commonly populates the human gut and urogenital tract, and is a major cause of infection-based mortality in neonatal infants and in elderly or immunocompromised adults. Nuclease A (GBS_NucA), a secreted DNA/RNA nuclease, serves as a virulence factor for S. agalactiae, facilitating bacterial evasion of the human innate immune response. GBS_NucA efficiently degrades the DNA matrix component of neutrophil extracellular traps (NETs), which attempt to kill and clear invading bacteria during the early stages of infection. In order to better understand the mechanisms of DNA substrate binding and catalysis of GBS_NucA, the high-resolution structure of a catalytically inactive mutant (H148G) was solved by X-ray crystallography. Several mutants on the surface of GBS_NucA which might influence DNA substrate binding and catalysis were generated and evaluated using an imidazole chemical rescue technique. While several of these mutants severely inhibited nuclease activity, two mutants (K146R and Q183A) exhibited significantly increased activity. These structural and biochemical studies have greatly increased our understanding of the mechanism of action of GBS_NucA in bacterial virulence and may serve as a foundation for the structure-based drug design of antibacterial compounds targeted to S. agalactiae.

Show MeSH
Related in: MedlinePlus