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Autoinducer-fluorophore conjugates enable FRET in LuxR proteins in vitro and in cells

Abstract

Cell-to-cell signaling, or quorum sensing (QS), in many Gram-negative bacteria is governed by small molecule signals (N-acyl-l-homoserine lactones, AHLs) and their cognate receptors (LuxR-type proteins). The mechanistic underpinnings of QS in these bacteria are severely limited due to the challenges of isolating and manipulating most LuxR-type proteins. Reports of quantitative direct-binding experiments on LuxR-type proteins are scarce, and robust and generalizable methods that provide such data are largely nonexistent. We report herein a Förster resonance energy transfer (FRET) assay that leverages (1) conserved tryptophans located in the LuxR-type protein ligand-binding site and synthetic fluorophore–AHL conjugates, and (2) isolation of the proteins bound to weak agonists. The FRET assay permits straightforward measurement of ligand-binding affinities with receptor—either in vitro or in cells—and was shown to be compatible with six LuxR-type proteins. These methods will advance fundamental investigations of LuxR-type protein mechanism and the development of small molecule QS modulators.

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Fig. 1: Design of an intrinsic tryptophan FRET assay for LuxR-type QS receptors.
Fig. 2: Synthesis and characterization of an AHL–fluorophore conjugate library.
Fig. 3: Development of an in vitro FRET assay.
Fig. 4: Development of an in-cell FRET assay.
Fig. 5: FRET assay data with receptors beyond QscR.
Fig. 6: Mechanistic FRET assays with various small molecules.

Data availability

The datasets generated and analyzed during this study are available from the corresponding author on request.

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Acknowledgements

Financial support for this work was provided by the National Institutes of Health (NIH) (grant no. R35 GM131817). M.J.S. was supported in part by the UW–Madison NIH Biotechnology Training Program (grant no. T32 GM008349) and a National Science Foundation (NSF) Graduate Research Fellowship (grant no. DGE-1747503). B.L.S. was supported in part by an NIH Ruth Kirschstein National Research Service Award (grant no. F32 AI138918). The authors made use of facilities at the UW–Madison including the Biophysics Instrumentation Facility (supported by the NSF (grant no. BIR-9512577) and NIH (grant no. S10 RR13790)) and at the UW–Madison Paul Bender Chemical Instrument Center.

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Contributions

H.E.B., M.E.B., M.J.S. and M.E.M. conceived and designed this project. M.J.S., M.A.M., M.E.M. and M.E.B. designed and synthesized the probe molecules. M.J.S., M.E.B., M.A.M. and E.E.S. performed all biological assays. M.J.S. and B.L.S. cloned plasmids. All authors participated in the analysis of the data. M.J.S., M.E.B., M.A.M. and H.E.B. wrote the manuscript, and all authors contributed to the editing of the manuscript.

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Correspondence to Helen E. Blackwell.

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Nature Chemical Biology thanks Martin Empting and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 FRET-probe library antagonism screen.

Each member of the FRET-probe library was assayed at 10 μM for antagonism with each receptor listed on the top of the heatmap in a heterologous reporter strain (see Supplementary Table 5 for strain details and main text) with the EC50 of the native ligand present (EC90 for SdiAse and SdiAec). Each receptor:FRET-probe pair has its own square in the heatmap. The heatmap is organized vertically by linker length (left of the heatmap), horizontally by receptor, and within a column by fluorophore (coumarin (CU) on the left and dansyl (DA) on the right). Receptor inhibition is normalized for each strain as the activity of the reporter with native ligand only (0%) and no ligand added (100%). See Supplementary Table 3 for native ligand identities and EC50 and EC90 concentrations. Each value is an average of two independent biological replicates, each with three technical replicates (a total of six independent replicates).

Extended Data Fig. 2 Cell-based reporter dose response curves for selected FRET-probes.

For each receptor, the native ligand (OdDHL and OOHL were used for the orphan receptors QscR and SdiA, respectively) and several FRET-probes identified as agonists (Fig. 2) were characterized by performing the cell-based transcriptional reporter assays at several concentrations to obtain dose-response plots. All assays were performed as three independent biological replicates, each with six technical replicates (a total of 18 independent replicates). Activation reported as the percentage (%) of activity normalized to the maximum activity of the native ligand (100% activation) and DMSO (0% activation). Error bars show s.d. Data were fit using GraphPad Prism software using a four parameter (varied slope) dose-response equation (see Supplementary Table 2 for parameter values from these fits).

Extended Data Fig. 3 Structures of small molecule modulators of QscR.

These compounds have been identified in previous studies by the Blackwell laboratory27,31. Compounds that are able to activate QscR to greater than 85–100% activity (relative to OdDHL) are categorized as agonists. Compounds able to reduce the activity of QscR in antagonism assays (with the EC50 of OdDHL present) are categorized as antagonists. Note, all the antagonists listed here are also able to partially activate QscR in agonism assay formats (10–70% relative to OdDHL).

Extended Data Fig. 4 Cell-based reporter assay dose-response curves for small molecule modulators of QscR.

Cell-based reporter assays were performed as described above for each compound (listed on x-axis of each plot) in an agonism format (data in black) and an antagonism format (data in red). Data was fit to either an activation or inhibition four parameter (varied slope) dose response equation in GraphPad Prism (model fits shown as lines). For compounds with a non-monotonic antagonism profile (OHHL, D6, C10, and S5)36, the upturn concentrations were not used in the model fit. All data points represent three independent biological replicates, each with three technical replicates; error bars indicate s.d.

Extended Data Fig. 5 In-cell FRET binding dose-response curves for select receptor:FRET-probe pairs.

In-cell FRET was performed as described above for each compound in a binding format (receptor and fluorophore listed above each plot). Data were fit to an activation four parameter (varied slope) dose-response equation in GraphPad Prism (model fits shown as lines and compiled in Supplementary Table 4). All data points represent at least two independent biological replicates, each with four technical replicates; error bars indicate s.d.

Extended Data Fig. 6 In-cell FRET displacement dose-response curves for select receptor:FRET-probe pairs.

In-cell FRET was performed as described above for each compound in a displacement format (receptor, fluorophore, and competing ligand listed above each plot). Data were fit to an inhibition four parameter (varied slope) dose-response equation in GraphPad Prism (model fits shown as lines and compiled in Supplementary Table 4). Fluorophore concentration was 10 μM in each case except for RhlR, for which 50 μM fluorophore was used. All data points represent at least two independent biological replicates, each with four technical replicates; error bars indicate s.d.

Extended Data Fig. 7 Activation of SdiAec by indole in a cell-based reporter assay.

An SdiAec cell-based reporter assay was performed as described above for indole in an agonism format. Data were fit to an activation four parameter (varied slope) dose-response equation in GraphPad Prism (model fit shown as a line). All data points represent three independent biological replicates, each with three technical replicates; error bars indicate s.d.

Extended Data Fig. 8 Representative QscR DSF assay results.

a, Representative DSF thermal melt profiles for QscR with no compound (DMSO, green), an agonist (OdDHL, black), and two representative examples of inhibitors (the partial agonist Q9, red; the non-monotonic partial agonist C10, blue). b, The first derivatives of the RFU data shown in part a.

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Supplementary Tables 1–7, Figs. 1–12 and Note 1.

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Styles, M.J., Boursier, M.E., McEwan, M.A. et al. Autoinducer-fluorophore conjugates enable FRET in LuxR proteins in vitro and in cells. Nat Chem Biol 18, 1115–1124 (2022). https://doi.org/10.1038/s41589-022-01089-1

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