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Molecular modeling reveals the novel inhibition mechanism and binding mode of three natural compounds to staphylococcal α-hemolysin.

Qiu J, Wang D, Zhang Y, Dong J, Wang J, Niu X - PLoS ONE (2013)

Bottom Line: This was completed using conventional Molecular Dynamics (MD) simulations.This novel inhibition mechanism has been confirmed by both the steered MD simulations and the experimental data obtained from a deoxycholate-induced oligomerization assay.This study can facilitate the design of new antibacterial drugs against S. aureus.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China.

ABSTRACT
α-Hemolysin (α-HL) is a self-assembling, channel-forming toxin that is produced as a soluble monomer by Staphylococcus aureus strains. Until now, α-HL has been a significant virulence target for the treatment of S. aureus infection. In our previous report, we demonstrated that some natural compounds could bind to α-HL. Due to the binding of those compounds, the conformational transition of α-HL from the monomer to the oligomer was blocked, which resulted in inhibition of the hemolytic activity of α-HL. However, these results have not indicated how the binding of the α-HL inhibitors influence the conformational transition of the whole protein during the oligomerization process. In this study, we found that three natural compounds, Oroxylin A 7-O-glucuronide (OLG), Oroxin A (ORA), and Oroxin B (ORB), when inhibiting the hemolytic activity of α-HL, could bind to the "stem" region of α-HL. This was completed using conventional Molecular Dynamics (MD) simulations. By interacting with the novel binding sites of α-HL, the ligands could form strong interactions with both sides of the binding cavity. The results of the principal component analysis (PCA) indicated that because of the inhibitors that bind to the "stem" region of α-HL, the conformational transition of α-HL from the monomer to the oligomer was restricted. This caused the inhibition of the hemolytic activity of α-HL. This novel inhibition mechanism has been confirmed by both the steered MD simulations and the experimental data obtained from a deoxycholate-induced oligomerization assay. This study can facilitate the design of new antibacterial drugs against S. aureus.

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OLG (A), ORA (B) and ORB (C) prevent the deoxycholate-induced oligomerization of α-HL.α-HL was treated with 5 mM deoxycholate in the presence or absence ligands. Following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis, the proteins were detected by silver staining. (A) Lane 1, WT-HL; lane 2, R104A-HL; lane 3, G126A-HL; lane 4, WT-HL plus 2 µg/ml of OLG; lane 5, R104A-HL plus 2 µg/ml of OLG; lane 6, G126A-HL plus 2 µg/ml of OLG. (B) Lane 1, WT-HL; lane 2, R104A-HL; lane 3, G126A-HL; lane 4, WT-HL plus 16 µg/ml of ORA; lane 5, R104A-HL plus 16 µg/ml of ORA; lane 6, G126A-HL plus 16 µg/ml of ORA. (C) Lane 1, WT-HL; lane 2, R104A-HL; lane 3, G126A-HL; lane 4, WT-HL plus 32 µg/ml of ORB; lane 5, R104A-HL plus 32 µg/ml of ORB; lane 6, G126A-HL plus 32 µg/ml of ORB.
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pone-0080197-g009: OLG (A), ORA (B) and ORB (C) prevent the deoxycholate-induced oligomerization of α-HL.α-HL was treated with 5 mM deoxycholate in the presence or absence ligands. Following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis, the proteins were detected by silver staining. (A) Lane 1, WT-HL; lane 2, R104A-HL; lane 3, G126A-HL; lane 4, WT-HL plus 2 µg/ml of OLG; lane 5, R104A-HL plus 2 µg/ml of OLG; lane 6, G126A-HL plus 2 µg/ml of OLG. (B) Lane 1, WT-HL; lane 2, R104A-HL; lane 3, G126A-HL; lane 4, WT-HL plus 16 µg/ml of ORA; lane 5, R104A-HL plus 16 µg/ml of ORA; lane 6, G126A-HL plus 16 µg/ml of ORA. (C) Lane 1, WT-HL; lane 2, R104A-HL; lane 3, G126A-HL; lane 4, WT-HL plus 32 µg/ml of ORB; lane 5, R104A-HL plus 32 µg/ml of ORB; lane 6, G126A-HL plus 32 µg/ml of ORB.

Mentions: This hypothesis is confirmed by the results of the experiment, as shown in Figure 9. Data from a deoxycholate-induced oligomeriazation assay [22] show that the site-directed mutagenesis of R104A and G126A has no influence on the assembly of the SDS-stable oligomer, α-HL7. However, the formation of α-HL7 was inhibited when treated with OLG (2 µg/ml), ORA (16 µg/ml) or ORB (32 µg/ml), which is in good agreement with the calculated results. Furthermore, this inhibitory effect was decreased with either of the two mutants due to the weaker affinities of the three ligands binding with α-HL in the protein-ligand complexes, as shown in Figure 9. These findings support the following inhibition mechanism: the binding of inhibitors to the “stem” domain blocks the conformational change from monomer to heptamer, thereby decreasing the lytic activity of α-HL.


Molecular modeling reveals the novel inhibition mechanism and binding mode of three natural compounds to staphylococcal α-hemolysin.

Qiu J, Wang D, Zhang Y, Dong J, Wang J, Niu X - PLoS ONE (2013)

OLG (A), ORA (B) and ORB (C) prevent the deoxycholate-induced oligomerization of α-HL.α-HL was treated with 5 mM deoxycholate in the presence or absence ligands. Following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis, the proteins were detected by silver staining. (A) Lane 1, WT-HL; lane 2, R104A-HL; lane 3, G126A-HL; lane 4, WT-HL plus 2 µg/ml of OLG; lane 5, R104A-HL plus 2 µg/ml of OLG; lane 6, G126A-HL plus 2 µg/ml of OLG. (B) Lane 1, WT-HL; lane 2, R104A-HL; lane 3, G126A-HL; lane 4, WT-HL plus 16 µg/ml of ORA; lane 5, R104A-HL plus 16 µg/ml of ORA; lane 6, G126A-HL plus 16 µg/ml of ORA. (C) Lane 1, WT-HL; lane 2, R104A-HL; lane 3, G126A-HL; lane 4, WT-HL plus 32 µg/ml of ORB; lane 5, R104A-HL plus 32 µg/ml of ORB; lane 6, G126A-HL plus 32 µg/ml of ORB.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842302&req=5

pone-0080197-g009: OLG (A), ORA (B) and ORB (C) prevent the deoxycholate-induced oligomerization of α-HL.α-HL was treated with 5 mM deoxycholate in the presence or absence ligands. Following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis, the proteins were detected by silver staining. (A) Lane 1, WT-HL; lane 2, R104A-HL; lane 3, G126A-HL; lane 4, WT-HL plus 2 µg/ml of OLG; lane 5, R104A-HL plus 2 µg/ml of OLG; lane 6, G126A-HL plus 2 µg/ml of OLG. (B) Lane 1, WT-HL; lane 2, R104A-HL; lane 3, G126A-HL; lane 4, WT-HL plus 16 µg/ml of ORA; lane 5, R104A-HL plus 16 µg/ml of ORA; lane 6, G126A-HL plus 16 µg/ml of ORA. (C) Lane 1, WT-HL; lane 2, R104A-HL; lane 3, G126A-HL; lane 4, WT-HL plus 32 µg/ml of ORB; lane 5, R104A-HL plus 32 µg/ml of ORB; lane 6, G126A-HL plus 32 µg/ml of ORB.
Mentions: This hypothesis is confirmed by the results of the experiment, as shown in Figure 9. Data from a deoxycholate-induced oligomeriazation assay [22] show that the site-directed mutagenesis of R104A and G126A has no influence on the assembly of the SDS-stable oligomer, α-HL7. However, the formation of α-HL7 was inhibited when treated with OLG (2 µg/ml), ORA (16 µg/ml) or ORB (32 µg/ml), which is in good agreement with the calculated results. Furthermore, this inhibitory effect was decreased with either of the two mutants due to the weaker affinities of the three ligands binding with α-HL in the protein-ligand complexes, as shown in Figure 9. These findings support the following inhibition mechanism: the binding of inhibitors to the “stem” domain blocks the conformational change from monomer to heptamer, thereby decreasing the lytic activity of α-HL.

Bottom Line: This was completed using conventional Molecular Dynamics (MD) simulations.This novel inhibition mechanism has been confirmed by both the steered MD simulations and the experimental data obtained from a deoxycholate-induced oligomerization assay.This study can facilitate the design of new antibacterial drugs against S. aureus.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, China.

ABSTRACT
α-Hemolysin (α-HL) is a self-assembling, channel-forming toxin that is produced as a soluble monomer by Staphylococcus aureus strains. Until now, α-HL has been a significant virulence target for the treatment of S. aureus infection. In our previous report, we demonstrated that some natural compounds could bind to α-HL. Due to the binding of those compounds, the conformational transition of α-HL from the monomer to the oligomer was blocked, which resulted in inhibition of the hemolytic activity of α-HL. However, these results have not indicated how the binding of the α-HL inhibitors influence the conformational transition of the whole protein during the oligomerization process. In this study, we found that three natural compounds, Oroxylin A 7-O-glucuronide (OLG), Oroxin A (ORA), and Oroxin B (ORB), when inhibiting the hemolytic activity of α-HL, could bind to the "stem" region of α-HL. This was completed using conventional Molecular Dynamics (MD) simulations. By interacting with the novel binding sites of α-HL, the ligands could form strong interactions with both sides of the binding cavity. The results of the principal component analysis (PCA) indicated that because of the inhibitors that bind to the "stem" region of α-HL, the conformational transition of α-HL from the monomer to the oligomer was restricted. This caused the inhibition of the hemolytic activity of α-HL. This novel inhibition mechanism has been confirmed by both the steered MD simulations and the experimental data obtained from a deoxycholate-induced oligomerization assay. This study can facilitate the design of new antibacterial drugs against S. aureus.

Show MeSH
Related in: MedlinePlus