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. 2011 Jul 19;108(29):12089-94.
doi: 10.1073/pnas.1103165108. Epub 2011 Jul 5.

Small-molecule inhibitor binding to an N-acyl-homoserine lactone synthase

Jiwoung Chung  1 , Eunhye Goo, Sangheon Yu, Okhee Choi, Jeehyun Lee, Jinwoo Kim, Hongsup Kim, Jun Igarashi, Hiroaki Suga, Jae Sun Moon, Ingyu Hwang, Sangkee Rhee
Affiliations

Small-molecule inhibitor binding to an N-acyl-homoserine lactone synthase

Jiwoung Chung et al. Proc Natl Acad Sci U S A. .

Abstract

Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In gram-negative bacteria, QS is often mediated by N-acyl-L-homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acyl-HSL synthases and their ligands. In the gram-negative bacterium Burkholderia glumae, QS controls virulence, motility, and protein secretion and is mediated by the binding of N-octanoyl-L-HSL (C8-HSL) to its cognate receptor, TofR. C8-HSL is synthesized by the acyl-HSL synthase TofI. In this study, we characterized two previously unknown QS inhibitors identified in a focused library of acyl-HSL analogs. Our functional and X-ray crystal structure analyses show that the first inhibitor, J8-C8, binds to TofI, occupying the binding site for the acyl chain of the TofI cognate substrate, acylated acyl-carrier protein. Moreover, the reaction byproduct, 5'-methylthioadenosine, independently binds to the binding site for a second substrate, S-adenosyl-L-methionine. Closer inspection of the mode of J8-C8 binding to TofI provides a likely molecular basis for the various substrate specificities of acyl-HSL synthases. The second inhibitor, E9C-3oxoC6, competitively inhibits C8-HSL binding to TofR. Our analysis of the binding of an inhibitor and a reaction byproduct to an acyl-HSL synthase may facilitate the design of a new class of QS-inhibiting therapeutic agents.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Identification of small molecules that interfere with QS in B. glumae. (A) Chemical structures of C8-HSL, J8-C8, and E9C-3oxoC6. (B) Inhibition of C8-HSL production in B. glumae BGR1 cells. (C) Inhibition of ahpF expression by various concentrations of J8-C8 and E9C-3oxoC6 in BGR1 cells. (D) Inhibition of toxoflavin production by various concentrations of E9C-3oxoC6 in B. glumae BGS2 (tofI::Ω) cells in the presence of 1 μM C8-HSL. Error bars represent SDs from triplicate experiments.
Fig. 2.
Fig. 2.
Molecular mechanisms of J8-C8 and E9C-3oxoC6 inhibitory activity. (A) Inhibition of C8-HSL synthesis by J8-C8 in BGR1 cells. (B) Inhibition of C8-HSL synthesis by MTA and synergistic inhibitory effect of J8-C8. The amount of C8-HSL produced for each treatment is shown relative to that produced for DMSO treatment alone (100%). (C) Toxoflavin inhibition in BGS2 cells in the presence of 12 μM E9C-3oxoC6 is relieved by increasing amounts of C8-HSL. Levels of toxoflavin production were measured in the presence of 12 μM E9C-3oxoC6 or 0.1% DMSO at 24 h and 32 h after the addition of C8-HSL. Error bars represent SDs from triplicate experiments.
Fig. 3.
Fig. 3.
Structure of TofI and the binding site of J8-C8. (A) The overall structure of apo-TofI(3MΔ) is shown with the putative pocket region, as defined by the program PocketPicker (23), indicated by dots. (B) The TofI(3MΔ)/J8-C8/MTA ternary complex is presented in an orientation identical to that in A. The bound J8-C8 is shown as a space-filling model, and the stabilized loop residues between α1 and β2 are shown in red. For clarity, a bound MTA in the ternary complex is not shown. (C) The J8-C8 binding site in the ternary complex is displayed with its neighboring residues and with the 2Fo–Fc electron density map (contoured at 0.9σ) for J8-C8. (D) Schematic diagram of the interactions between TofI(3MΔ) and J8-C8 in the ternary complex. Enzyme residues involved in direct hydrogen bonds are indicated in red with interatomic distances (Å). Residues involved in van der Waals interactions within 4.0 Å of the acyl chain or ring moiety of J8-C8 are shown in green and black, respectively. A water molecule is indicated with a red circle. (E) The pocket-shaped binding site of J8-C8 in the ternary complex is shown as a surface model. This view is rotated by ∼180° from that shown in C.
Fig. 4.
Fig. 4.
Structural and functional features of the ternary complex. (A) The overall structure of the ternary complex. J8-C8 and MTA are represented by space-filling models, and the stabilized loop residues between α1 and β2 are shown in red. (B) Close-up molecular surface representation of the J8-C8 and MTA binding sites separated by Phe146 and Trp33. (C) Close-up view of the MTA-binding region and the nearby interacting residues. The Fo–Fc electron density map for MTA is contoured at 3.5σ. (D) Schematic diagram of the interactions between bound MTA and residues in its binding site. Residues participating in hydrogen bonds (red) and in van der Waals interactions (green) are shown. (E) Detection of acyl-HSLs produced by various TofI enzymes. Unlike the other enzymes, the S103G and E101Q mutants contain additional mutations shown in TofI(3MΔ) to express the mutant protein as a soluble form. Details of the thin-layer chromatographic analysis are provided in SI Appendix, Fig. S1B.
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