Detailed inhibitor-induced inhibition activation mechanism of α-HL.

<p>The inhibitors OLG, ORA and ORB can bind to the “stem” domain of α-HL by interactions with Tyr102/Pro103/Arg104/Tyr112 and Gly126. The ligands can form strong interactions with both sides of the binding cavity. Because of the ligands binding with the “stem” domain of α-HL, the conformational transition of α-HL from the monomeric α-HL to the oligomer was restricted, which leads to the inhibition of the hemolytic activity of α-HL.</p

Collective motions obtained by principal component analysis on the simulation trajectory.

<p>(A) and (B) Motions corresponding to PC1 and PC2 of the unliganded α-HL, which account for 68.7 and 19.8% of the total movements, respectively. (C) and (D) Motions corresponding to PC1 and PC2 of the α-HL-OLG complex, which account for 65.4% and 18.7% of the total movements, respectively.</p

The RMS fluctuations of the whole residues in the complex and free α-HL.

<p>The region of the protein backbone exhibits different fluctuations, which are dependent on the local environment (binding with inhibitors), specifically residues 100–150. The region is highlighted with gray bars.</p

The values of the binding free energy (<i>ΔGbind</i>) and the number of binding sites (n) of the α-HL-inhibitor systems based on the results from the fluorescence-quenching method.

<p>The values of the binding free energy (<i>ΔGbind</i>) and the number of binding sites (n) of the α-HL-inhibitor systems based on the results from the fluorescence-quenching method.</p

OLG (A), ORA (B) and ORB (C) prevent the deoxycholate-induced oligomerization of α-HL.

<p>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.</p

Summed energies of interaction between the inhibitors and α-HL.

<p>The histogram shows the van der Waals, electrostatic, salvation and total contributions for the complexes.</p

The calculated energy components, the binding free energy (kcal/mol) of OLG, ORA, and ORB binding to the active site of α-HL.

<p>The calculated energy components, the binding free energy (kcal/mol) of OLG, ORA, and ORB binding to the active site of α-HL.</p

(A) Overview of the α-HL: compound complexes based on MD simulation.

<p>The arrows indicate the predicted binding mode of oroxylin A 7-O-glucuronide (OLG) with α-HL (B), oroxin A (ORA) with α-HL (C), and oroxin B (ORB) with α-HL (D). The shadow region represents the left region (L) and right region (R) of the binding cavity.</p

Hemolytic activity of α-HL after treating with increasing concentrations (µg/ml) of OLG, ORA and ORB. “+” indicates the positive control while “-” indicates the negative control.

<p>Hemolytic activity of α-HL after treating with increasing concentrations (µg/ml) of OLG, ORA and ORB. “+” indicates the positive control while “-” indicates the negative control.</p

Hemolytic activities of α-hemolysin produced by <i>S. aureus</i> cultured with graded subinhibitory concentrations of thymol.

<p><i>^a</i>The drug-free culture supernatants served as 100% hemolysis.</p><p>NO. means no hemolytic activity was observed.</p><p>Values represent the mean and standard error of three independent experiments.</p>*<p>, <i>p</i><0.05 and</p>**<p>, <i>p</i><0.01 compared to the corresponding control.</p