Unraveling the contributions of hydrogen-bonding interactions to the activity of native and non-native ligands in the quorum-sensing receptor LasR

Gerdt, Joseph P.; McInnis, Christine E.; Schell, Trevor L.; Blackwell, Helen E.

doi:10.1039/c4ob02252a

Cited by 26 publications

(31 citation statements)

References 68 publications

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“…2D). Our results join previous findings suggesting that autoinducer acyl side chain length is particularly important in dictating the stimulation of LasR activity (36) and recent work cataloging hydrogen-bonding interactions in the LasR LBD critical for LasR activation and inhibition (37,38).…”

Section: Impact Of Oxidative Stress On the Lasr Ligand Binding Domainsupporting

confidence: 89%

Molecular Insights into the Impact of Oxidative Stress on the Quorum-Sensing Regulator Protein LasR

Kafle¹,

Amoh²,

Reaves³

et al. 2016

Journal of Biological Chemistry

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The LasR regulator protein functions at the top of the Pseudomonas aeruginosa quorum-sensing hierarchy and is implicated in promoting bacterial virulence. Of note is recent evidence that this transcription factor may also respond to oxidative stress. Here, all cysteines in LasR were inspected to deduce their redox sensitivity and to probe the connection between stress response and LasR activity using purified LasR and individual LasR domains. Cys 79 in the ligand binding domain of LasR appears to be important for ligand recognition and folding of this domain to potentiate DNA binding but does not seem to be sensitive to oxidative stress when bound to its native ligand. Two cysteines in the DNA binding domain of LasR do form a disulfide bond when treated with hydrogen peroxide, and formation of this Cys 201 -Cys 203 disulfide bond appears to disrupt the DNA binding activity of the transcription factor. Mutagenesis of either of these cysteines leads to expression of a protein that no longer binds DNA. A cell-based reporter assay linking LasR function with ␤-galactosidase activity gave results consistent with those obtained with purified LasR. This work provides a possible mechanism for oxidative stress response by LasR and indicates that multiple cysteines within the protein may prove to be useful targets for disabling its activity.The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen that establishes chronic infections in immunocompromised patients, with persistent pulmonary colonization in individuals with cystic fibrosis (1-3). Like many bacteria, P. aeruginosa employs quorumsensing (QS) 4 machinery to communicate local population density through the exchange of self-produced signaling molecules (4). QS contributes to the development of acute infections by coordinating the production of multiple virulence factors, including elastase and pyocyanin, and the formation of biofilms (5-7). QS in P. aeruginosa is largely mediated by N-acyl L-homoserine lactone autoinducer molecules, biosynthesized by LuxI-type synthases, and detected by LuxR-type regulator proteins (8). The two major systems responsive to acylhomoserine lactones (AHLs) in P. aeruginosa are LasI/LasR and RhlI/RhlR. LasI and RhlI are LuxI-type synthases, whereas LasR and RhlR are LuxR-type regulatory proteins. These AHL-dependent systems are also closely connected to signaling governed by 2-alkyl-4-quinolones in P. aeruginosa (9).The regulator protein LasR, with its cognate synthase LasI, functions at the top of the P. aeruginosa quorum-sensing hierarchy (10). Like other LuxR-type proteins, this transcription factor is composed of two domains (11). The amino-terminal ligand binding domain (LBD) specifically recognizes its autoinducer, N-(3-oxo-dodecanoyl)-L-homoserine lactone (3O-C 12 -HSL), promoting protein folding and dimerization, whereas the DNA binding domain (DBD) recognizes target sites to activate downstream gene expression. There has been significant interest in understanding LasR, as its inhibition represents a...

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Section: Impact Of Oxidative Stress On the Lasr Ligand Binding Domainsupporting

confidence: 89%

Molecular Insights into the Impact of Oxidative Stress on the Quorum-Sensing Regulator Protein LasR

Kafle¹,

Amoh²,

Reaves³

et al. 2016

Journal of Biological Chemistry

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“…Subsequent mutational analyses revealed that modification of a single LasR residue, Trp60, that hydrogen bonds with the carbonyl moiety of the ligand lactone ring can convert an agonist into an antagonist and vice versa 42 . Consistent with these findings, substituting the lactone head group on the native AI with a phenyl head group converts the ligand into an antagonist 42,43 .…”

supporting

confidence: 56%

An Autoinducer Analogue Reveals an Alternative Mode of Ligand Binding for the LasR Quorum-Sensing Receptor

et al. 2019

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Bacteria use a cell-cell communication process called quorum sensing to coordinate collective behaviors. Quorum sensing relies on production and group-wide detection of extracellular signal molecules called autoinducers. Here, we probe the activity of the Pseudomonas aeruginosa LasR quorum-sensing receptor using synthetic agonists based on the structure of the native homoserine lactone autoinducer. The synthetic compounds range from low to high potency, and agonist activity tracks with the ability of the agonist to stabilize the LasR protein. Structural analyses of the LasR ligand binding domain complexed with representative synthetic agonists reveal two modes of ligand binding, one mimicking the canonical autoinducer binding arrangement and the other with the lactone head group rotated approximately 150°. Iterative mutagenesis combined with chemical synthesis reveals the amino acid residues and the chemical moieties, respectively, that are key to enabling each mode of binding. Simultaneous alteration of LasR residues Thr75, Tyr93, and Ala127 converts low-potency compounds into high-potency compounds and converts ligands that are nearly inactive into low-potency compounds. These results show that the LasR binding pocket displays significant flexibility in accommodating different ligands. The ability of LasR to bind ligands in different conformations, and in so doing, alter their potency as agonists, could explain the difficulties that have been encountered in the development of competitive LasR inhibitors.

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“…Among the set of mutants that we made, we identified one residue of interest: LasR S129. Previous work demonstrated that alteration of serine to alanine at this position transformed some LasR activators into inhibitors, and vice versa (32,33). Moreover, the LasR LBD structure suggests that S129 is part of the network that interacts with the ligand acyl chain (29).…”

Section: Resultsmentioning

confidence: 93%

Structural determinants driving homoserine lactone ligand selection in the Pseudomonas aeruginosa LasR quorum-sensing receptor

McCready

Paczkowski

Henke³

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Quorum sensing is a cell-cell communication process that bacteria use to orchestrate group behaviors. Quorum sensing is mediated by signal molecules called autoinducers. Autoinducers are often structurally similar, raising questions concerning how bacteria distinguish among them. Here, we use the Pseudomonas aeruginosa LasR quorum-sensing receptor to explore signal discrimination. The cognate autoinducer, 3OC 12 homoserine lactone (3OC 12 HSL), is a more potent activator of LasR than other homoserine lactones. However, other homoserine lactones can elicit LasR-dependent quorum-sensing responses, showing that LasR displays ligand promiscuity. We identify mutants that alter which homoserine lactones LasR detects. Substitution at residue S129 decreases the LasR response to 3OC 12 HSL, while enhancing discrimination against noncognate autoinducers. Conversely, the LasR L130F mutation increases the potency of 3OC 12 HSL and other homoserine lactones. We solve crystal structures of LasR ligand-binding domains complexed with noncognate autoinducers. Comparison with existing structures reveals that ligand selectivity/ sensitivity is mediated by a flexible loop near the ligand-binding site. We show that LasR variants with modified ligand preferences exhibit altered quorum-sensing responses to autoinducers in vivo. We suggest that possessing some ligand promiscuity endows LasR with the ability to optimally regulate quorum-sensing traits.crystal structure | LasR | Pseudomonas aeruginosa | quorum sensing | homoserine lactone

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Unraveling the contributions of hydrogen-bonding interactions to the activity of native and non-native ligands in the quorum-sensing receptor LasR

Cited by 26 publications

References 68 publications

Molecular Insights into the Impact of Oxidative Stress on the Quorum-Sensing Regulator Protein LasR

Molecular Insights into the Impact of Oxidative Stress on the Quorum-Sensing Regulator Protein LasR

An Autoinducer Analogue Reveals an Alternative Mode of Ligand Binding for the LasR Quorum-Sensing Receptor

Structural determinants driving homoserine lactone ligand selection in the Pseudomonas aeruginosa LasR quorum-sensing receptor

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