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Molecular basis for control of antibiotic production by a bacterial hormone


Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture1. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear2. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR3. Here we report the X-ray crystal structure of an MmfR–AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR–operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics.

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Fig. 1: Classes of actinobacterial hormone that induce antibiotic production by binding TFTRs and proposed mechanism for regulation of methylenomycin A biosynthesis by AHFCAs.
Fig. 2: Structures of apo-MmfR and the MmfR–MMF2 complex determined by X-ray crystallography, and the cryo-EM structure of the MmfR–MARE1 complex.
Fig. 3: Insights into the mechanism of signal transduction in MmfR and other ArpA-like TFTRs.

Data availability

The atomic coordinates have been deposited in the Protein Data Bank for apo and liganded MmfR under the accession numbers 7KY1 and 6SRN, respectively. The three-dimensional cryo-EM map of the protein–DNA complex has been deposited in the Electron Microscopy Data Bank (EMDB) under the accession number EMD-20781. This EMDB entry includes raw half maps, the B-factor sharpened map, and the mask used for refinement and sharpening. Source data are provided with this paper.


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C.C. acknowledges support of this work by a University Research Fellowship from The Royal Society (UF090255) and by grant BB/M022765/1 from the UK Biotechnology and Biological Sciences Research Council (BBSRC). This work was also supported by BBSRC grant BB/M017982/1 (to G.L.C. and C.C.). G.L.C. is grateful to the Monash–Warwick Alliance (postdoctoral fellowship to H.B.), the University of Warwick (Chancellor’s International Scholarship to S.Z., Warwick Collaborative Postgraduate Research Scholarship to N.M. and Institute of Advanced Study Postdoctoral Research Fellowship to P.K.S.) and the ARC Centre of Excellence in Advanced Molecular Imaging for support of this work. Crystallographic data were collected at beam lines IO3, IO4 and I24 at Diamond Light Source, UK and we acknowledge the support of the beam line scientists. We thank C. J. Lupton for assisting with instrument set-up for the fluorescence anisotropy measurements and I. Prokes for assistance with acquiring the NMR data.

Author information

Authors and Affiliations



C.C. designed the expression vector for MmfR. S.Z., K.M.S., G.L.C. and C.C. designed site-directed mutagenesis experiments. S.Z., P.J.H., G.L.C. and C.C. designed the EMSA experiments. H.B. designed the FA experiments. S.Z., N.M. and G.L.C. designed the synthesis of the AHFCAs, analogues and SCB1. N.M., G.L.C. and C.C. designed the experiments to assess methylenomycin induction in vivo using the library of AHFCAs and analogues. H.B., M.J.B., H.V., M.J.C. and G.L.C. designed cryo-EM experiments. S.Z., H.B., P.J.H., D.R. and C.C. overproduced and purified recombinant MmfR. S.Z. and L.S. performed protein MS analyses. S.Z. and K.M.S. created the MmfR mutants. S.Z. and N.M. synthesized the library of AHFCAs, analogues and SCB1. S.Z., P.J.H. and C.C. performed EMSAs. H.B. performed FA measurements. N.M. and C.C. investigated the in vivo activity of AHFCAs and analogues. D.R., P.K.S. and V.F. prepared samples for protein crystallization, and D.R. and V.F. collected X-ray diffraction data. H.B. prepared samples for cryo-EM analysis and H.V. collected cryo-EM data. S.Z., H.B., N.M., P.J.H., G.L.C. and C.C. analysed the results of EMSAs. H.B., M.J.C. and G.L.C. analysed the results of FA measurements. D.R., H.B. and V.F. processed X-ray diffraction data and solved protein structures. S.Z., D.R., H.B., L.M.A., V.F., G.L.C. and C.C. analysed the X-ray crystal structures. H.B., M.J.B. and H.V. processed cryo-EM data. H.B., M.J.B., M.J.C. and G.L.C. analysed the cryo-EM structure. S.Z., H.B., N.M., L.M.A., G.L.C. and C.C. wrote the manuscript with input from the other authors.

Corresponding authors

Correspondence to Gregory L. Challis or Christophe Corre.

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Competing interests

G.L.C. is a co-director of Erebagen Ltd; the other authors declare no competing interests.

Additional information

Peer review information Nature thanks Robert Landick, Justin Nodwell and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Comparison of the mode of DNA and ligand binding, and mechanism of ligand-induced DNA release in TFTRs that regulate the expression of genes for antibiotic resistance and biosynthesis.

For clarity, only α-helices 1–3 forming the DBD in both repressor types, α-helices 4 and 6 in the HBD of the biosynthesis repressors and α-helices 4, 6, 9 and 10 in the antibiotic-binding domain (ABD) of the resistance repressors are shown in the schematics. The direction of movement of the DBD upon ligand binding is highlighted with an arrow. The distance between α-helices 3 and 3′ before and after DNA release and the distance between α-helix 1 and the ligand after release are given in Å. The structures of the antibiotics or hormones that act as ligands are shown and the name of the TFTR that each bind to is given in parentheses. a, TetR and SimR are examples of TFTRs that regulate antibiotic resistance. A single homodimer of these proteins binds to the operator and the ligand-binding site in the ABD is formed by residues from both subunits. b, MmfR as an example of a TFTR that regulates antibiotic biosynthesis. Two homodimers bind to the operator and the ligand-binding site in the HBD is formed by residues from only a single subunit. PDB entries for the DNA-bound form of TetR and SimR can be found under accession numbers 1QPI and 3QZL, respectively and their liganded entries under 2TRT and 2Y30, respectively.

Extended Data Fig. 2 Confirmation of the purity and identity of recombinant MmfR and in vivo assay for induction of methylenomycin production in S. coelicolor.

a, Left, analysis of purified recombinant His6-MmfR by SDS–PAGE. MWM, molecular weight marker. Right, mass spectrometry analysis: measured (top) and deconvoluted (bottom) mass spectra of His6-MmfR. Calculated mass, 27,835.5 Da. b, In vivo assay for induction of methylenomycin production upon addition of increasing amounts of MMF1 to growing mycelia of the MMF non-producing strain S. coelicolor W81. Methylenomycin production was detected by growth inhibition of the methylenomycin-sensitive strain S. coelicolor M145.

Extended Data Fig. 3 EMSA data for release of wild-type and mutant MmfR from the mmyB–mmyY and mmfL–mmfR intergenic regions in the presence of increasing quantities of the MMFs, MMF analogues and SCB1.

a, Interaction of MmfR with DNA fragments corresponding to the mmyBmmyY intergenic region (230 bp) in response to increasing amounts of MMFs. b, Interaction of MmfR with DNA fragments corresponding to the mmfLmmfR intergenic region (194 bp) in response to increasing amounts of MMFs. c, Interaction of MmfR with the DNA fragments corresponding to the mmfLmmfR intergenic region (194 bp) in response to increasing amounts of SCB1. d, EMSAs showing that Y85 and Q130 of MmfR have an important role in hormone-induced DNA release. Approximately ten times the quantity of MMF1 is required to release the Y85F and Q130E mutants of MmfR from the mmfLmmfR intergenic region (194 bp) than the wild-type protein. e, Interaction of MmfR with the DNA fragments corresponding to the mmfLmmfR intergenic region (194 bp) in response to increasing amounts of synthetic MMF analogues. Lane 1, isolated DNA fragments (0.1 pmol); lane 2, DNA fragments (0.1 pmol) mixed with protein (1.8 pmol). For a, b and d, lanes 3 to 9 show addition of increasing quantities of MMFs (0.8, 4, 8, 14, 20, 40 and 100 nmol respectively) to the protein–DNA complexes. For c, lanes 3 to 7 show addition of increasing quantities of racemic SCB1 (0.8, 8, 20, 40 and 400 nmol respectively) to the DNA–protein complexes. For e, lanes 3 to 6 show addition of increasing quantities of MMF analogues (0.8, 8, 20 and 100 nmol respectively) to the DNA–protein complexes. At least two independent technical replicates of each EMSA were conducted and in all cases similar results were obtained. For gel source data, see Supplementary Fig. 1a–d.

Extended Data Fig. 4 Results of fluorescence anisotropy measurements and EMSAs showing that MmfR binds MAREs in the mmfL–mmfR and mmyB–mmyY intergenic regions and is released from the MAREs by the MMFs.

a, Fluorescence anisotropy plots for binding of MmfR to DNA duplexes containing MARE1 and MARE2. Data points are the mean of three independent technical replicates (n = 3) and error bars represent the standard deviations from the mean. The Kd (nM) and Hill coefficient (ƞ) calculated from each dataset are shown. The structure of the DNA duplex used in each experiment is shown below the plot. b, Confirmation from EMSAs that the five naturally occurring MMFs are able to release MmfR from the mmfLmmfR and mmyBmmyY intergenic regions, and EC50 values calculated from fluorescence anisotropy measurements for the release of MmfR from DNA duplexes containing MARE1 and MARE2.

Source data

Extended Data Fig. 5 Synthetic routes to MMFs and analogues and SCB1.

a, Synthetic route to SCB1. b, General synthetic route to MMFs and most analogues. c, Synthetic route for MMF analogue 1 lacking a 2-alkyl group.

Extended Data Fig. 6 Electron density map for MMF2 bound to MmfR, comparison of the overall fold of the MmfR and CprB monomers and X-ray crystal structures of TFTR–operator complexes.

a, SIGMAA-weighted mFo − ΔFc electron density omit map for MMF2 bound to MmfR in mesh representation contoured at the 5σ level. b, Overall fold of the MmfR monomer. c, Overall fold of the CprB monomer (PDB ID: 4PXI). Both structures are colour-ramped from blue to red from the N to the C terminus. d, X-ray crystal structures of TFTRs that bind as pairs of homodimers in complex with their operators. PDB IDs are as follows: 6EN8 (SaFadR), 6C31 (Rv0078), 4JL3 (Ms6564), 5GPC (FadR), 1JT0 (QacR), 4I6Z (TM1030), 2YVH (CgmR), 4PXI (CprB), 5VL9 (EilR) and 4GCT (SlmA). e, X-ray crystal structures of TFTRs that bind as single homodimers in complex with their operators. PDB IDs are as follows: 1QPI (TetR), 5DY0 (AmtR), 3LSP (DesT), 5UA1 (KstR), 3ZQL (SimR), 5K7Z (AibR), 3VOK (HrtR) and 5YEJ (BioQ). f, Side view of the QacR–operator and CprB–operator complexes highlighting the obtuse angle between the planes that bisect the monomers in each homodimer.

Extended Data Fig. 7 Data quality, overall view of model and cryo-EM map fit for the MmfR–MARE1 complex.

a, RELION-corrected Fourier shell correlation (FSC) curve of the protein–DNA complex map. The inset shows the angular distribution of the particle projections. The length of the projection is a direct measure of the number of assigned particles in each direction. b, Model construction by fitting the coordinates for the MmfR–MMF2 complex (pink and blue cartoon) into the cryo-EM density map. Different views of the cryo-EM density maps with the protein–DNA complex modelled into it are shown to the right. c, Magnified view of the DBD showing how differently it is oriented in the MmfR–MMF2 complex compared to the MmfR–MARE1 complex. d, Local resolution values projected onto the experimental density map.

Extended Data Fig. 8 Sequence alignment of MmfR with other ArpA-like TFTRs and structural mapping of conserved residues.

a, Multiple sequence alignment of TFTRs of known hormone specificity. Amino acids showing a high degree of conservation are coloured yellow, whereas those showing a low degree of conservation are coloured grey. Highly conserved residues thought to be involved in the signal transmission from α-helices 4 and 6, through α-helix 1 to α-helices 2 and 3 in all TFTRs are marked ¥. The highly conserved residues and residue F182, which is only conserved in AHFCA-binding TFTRs, lining the hydrophobic pocket of the HBD are indicated with * and £, respectively. The Y85 and Q130 residues, universally conserved in AHFCA-binding TFTRs, are marked §. Protein names are coloured according to the type of ligand each TFTR responds to: cyan, AHFCAs; red, GBLs; purple, AHMBs; blue, ABs; pink, unknown. b, Mapping of residues showing a high (yellow) and low (grey) degree of conservation onto the structure of the MmfR–MMF2 complex.

Extended Data Table 1 Induction of methylenomycin production by MMFs and analogues, and SCB1 in S. coelicolor
Extended Data Table 2 Results of EMSA and fluorescence anisotropy experiments to investigate the role of hormone functional groups in DNA release using an MMF analogue library

Supplementary information

Supplementary Information

This file contains the Supplementary Methods and Supplementary Tables 1-4, 2 Supplementary Equations and Supplementary References.

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Supplementary Figures

This file contains Supplementary Figs 1-35.

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Zhou, S., Bhukya, H., Malet, N. et al. Molecular basis for control of antibiotic production by a bacterial hormone. Nature 590, 463–467 (2021).

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