Structural and Biochemical Studies of Non-native Agonists of the LasR Quorum-Sensing Receptor Reveal an L3 Loop “Out” Conformation for LasR

Abstract

SUMMARY Chemical strategies to block quorum sensing (QS) could provide a route to attenuate virulence in bacterial pathogens. Considerable research has focused on this approach in Pseudomonas aeruginosa, which uses the LuxR-type receptor LasR to regulate much of its QS network. Non-native ligands that antagonize LasR have been developed, yet we have little understanding of the mode by which these compounds interact with LasR and alter its function, as the receptor is unstable in their presence. Herein, we report an approach to circumvent this challenge though the study of a series of synthetic LasR agonists with varying levels of potency. Structural investigations of these ligands with the LasR ligand-binding domain reveal that certain agonists can enforce a conformation that deviates from that observed for other, often more potent agonists. These results, when combined with cell-based and biophysical analyses, suggest a functional model for LasR that could guide future ligand design.

Non-native autoinducer analogs capable of modulating the SdiA quorum sensing receptor in Salmonella enterica serovar Typhimurium

“…The sterically larger tails of the SdiA antagonists uncovered here could prevent the closing of the SdiA binding pocket and the transition to the more active state. While additional studies are clearly necessary to support this hypothesis, it is congruent with the SdiA structural data, the observation that 1-octanoyl- rac -glycerol ( 23 ) has no activity in the SdiA reporter assay, and recently reported data for LasR suggestive of a closed ligand-binding site for maximal activation [ 65 ].…”

Attenuation of Pseudomonas aeruginosa Quorum Sensing by Natural Products: Virtual Screening, Evaluation and Biomolecular Interactions

“…These results suggested that the interactions of the acyl side chain with the loop region promotes increased protein stability, in turn activates LasR. It is notable that the packing of Tyr 47 against the acyl chain protects the LBD from the bulk solvent and alterations at L3 loop region, leading to protein instability [12]. Also, LasR is agonized by ligands that were able to form H-bonds with Trp60 and Asp73 residues, and antagonized if they interacted with Tyr 47.…”

“…Since Tyr-47 is positioned in the L3 loop region which covers the LBD, the Tyr-47 interaction in the absence of the autoinducer would result in loop flexibility, and subsequent exposure of the hidden hydrophobic residues in LBD resulting in protein aggregation [35]. Compounds that induce inward moment of L3 loop with the bond length of 3.5-3.9Å found to be potential LasR inhibitors [12]. In MST we could not find any misfolding of LasR since the fluorescent dye, its interaction with protein and buffer system are entirely different from Thermal shift assay system.…”

“…These systems are well-organized with LasI/R at the top of the hierarchy in the cascade. LasR consists of two independently folded domains: A larger ligand binding domain (LBD) at the amino-terminal and a smaller DNA-binding domain (DBD) at the carboxy-terminal [12]. When N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL), an AI of LasI/R system, interacts with LasR, it activates many downstream genes including the lasI synthase, and leads to the production of 3OC12-HSL.…”

Virtual Screening of FDA-Approved Drugs against LasR of Pseudomonas aeruginosa for Antibiofilm Potential

“…Binding of homoserine lactone autoinducer promotes the dimerization of two LasR subunits. The resulting ligand-bound homodimer binds target DNA to activate gene transcription [13]. The structure of LasR-LBD (Protein Data Bank (PDB) code: 6D6A) with the known ligand, TP1, is shown in Figure 1.…”