The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors

Abstract

The function of LuxR homologues as quorum sensors is mediated by the binding of N-acyl-l-homoserine lactone (AHL) signal molecules to the N-terminal receptor site of the proteins. In this study, site-directed mutagenesis was carried out of the amino acid residues comprising the receptor site of LuxR from Vibrio fischeri, and the ability of the L42A, L42S, Y62F, W66F, D79N, W94D, V109D, V109T and M135A LuxR mutant proteins to activate green fluorescent protein expression from a PluxI promoter was measured. X-ray crystallographic studies of the LuxR homologue TraR indicated that residues Y53 and W57 form hydrogen bonds to the 1-carbonyl group and the ring carbonyl group, respectively, of the cognate AHL signal. Based on the activity and signal specificity of the LuxR mutant proteins, and on molecular modelling, a model is suggested in which Y62 (corresponding to Y53 in TraR) forms a hydrogen bond with the ring carbonyl group rather than the 1-carbonyl group, while W66 (corresponding to W57 in TraR) forms a hydrogen bond to the 1-carbonyl group. This flips the position of the acyl side chain in the LuxR/signal molecule complex compared to the TraR/signal molecule complex. Halogenated furanones from the marine alga Delisea pulchra and the synthetic signal analogue N-(sulfanylacetyl)-l-homoserine lactone can block quorum sensing. The LuxR mutant proteins were insensitive to inhibition by N-(propylsulfanylacetyl)-l-homoserine lactone. In contrast, the mutations had only a minor effect on the sensitivity of the proteins to halogenated furanones, and the data strongly suggest that these compounds do not compete in a ‘classic’ way with N-3-oxohexanoyl-l-homoserine lactone for the binding site. Based on modelling and experimental data it is suggested that these compounds bind in a non-agonist fashion.

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…Evidence of direct binding to either type of receptor comes from genetic and biochemical studies indicating that furanones bind at or near signal-binding pockets but interact with them in a significantly differently way than agonistic molecules. Illustrating this idea, LuxR mutant proteins that interfere with C8HSL binding impacted sensitivity to furanone treatments only slightly, indicating different binding modes for the two types of ligands (258). However, these modest inhibitory effects were reversed with further C8HSL addition, supporting the notion of a competitive interaction for the binding site.…”

“…However, these modest inhibitory effects were reversed with further C8HSL addition, supporting the notion of a competitive interaction for the binding site. Separately, furanones have been observed to enhance cellular turnover of LuxR proteins (237,258), counteracting the stabilizing effects provided by AHLs (259). Taking the data together, furanones appear not to inhibit LuxR activation by a "classic" competition mechanism, but rather they appear to engage LuxR at sites that are somewhat different from those used in AHL interactions and thus cause protein instability.…”

Quorum quenching quandary: resistance to antivirulence compounds

“…Under these conditions, there will be selection pressure to evolve resistance against the compounds that block QS, and it has been theorized, but not shown, that resistance may arise (Defoirdt et al, 2010). Note that previously mutations to the QS response regulator LuxR were generated in Escherichia coli, which altered both the binding of the natural ligand as well as that of quorum quenching (QQ) compounds, but resistance was not investigated and was predicted not to occur (Koch et al, 2005).…”

Directed evolution of the autoinducer selectivity of <i>Vibrio fischeri</i> LuxR

“…Y62 is highly conserved among LuxR family members (e.g., Whitehead et al, 2001) and is believed to form a hydrogen bond with the ring carbonyl group in acyl-HSLs (e.g., Koch et al, 2005). Previously, Koch et al (2005) claimed that Y62F abolished the binding of both 3OC6HSL and 3OC8HSL.…”

“…Previously, Koch et al (2005) claimed that Y62F abolished the binding of both 3OC6HSL and 3OC8HSL. We assume that Y62H also eliminated the hydrogen bond with the ring carbonyl group, thereby desensitizing LuxR both to 3OC8HSL and 3OC6-HSL.…”