Abstract
Antibiotics that target Gram-negative bacteria in new ways are needed to resolve the antimicrobial resistance crisis1,2,3. Gram-negative bacteria are protected by an additional outer membrane, rendering proteins on the cell surface attractive drug targets4,5. The natural compound darobactin targets the bacterial insertase BamA6—the central unit of the essential BAM complex, which facilitates the folding and insertion of outer membrane proteins7,8,9,10,11,12,13. BamA lacks a typical catalytic centre, and it is not obvious how a small molecule such as darobactin might inhibit its function. Here we resolve the mode of action of darobactin at the atomic level using a combination of cryo-electron microscopy, X-ray crystallography, native mass spectrometry, in vivo experiments and molecular dynamics simulations. Two cyclizations pre-organize the darobactin peptide in a rigid β-strand conformation. This creates a mimic of the recognition signal of native substrates with a superior ability to bind to the lateral gate of BamA. Upon binding, darobactin replaces a lipid molecule from the lateral gate to use the membrane environment as an extended binding pocket. Because the interaction between darobactin and BamA is largely mediated by backbone contacts, it is particularly robust against potential resistance mutations. Our results identify the lateral gate as a functional hotspot in BamA and will allow the rational design of antibiotics that target this bacterial Achilles heel.
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Data availability
The data that support the findings of this study are available from the corresponding author upon request. The atomic coordinates have been deposited in the RCSB Protein Data Bank and are available under the accession codes 7NRE and 7NRF. The cryo-EM map has been deposited in the Protein Data Bank under accession code 7NRI and EMDB accession code 12546. Mass spectrometry data have been deposited in figshare at https://doi.org/10.6084/m9.figshare.12179784. For the study, data were retrieved from the OMPdb (release Dec 1, 2020). Source data are provided with this paper.
Code availability
The codes developed for β-signal analysis have been deposited at https://github.com/hiller-lab/kaur-jakob-2021.
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Acknowledgements
We thank H. Bernstein and C. Bieniossek for expression plasmids, and T. Sharpe, T. Müntener and the Biozentrum Bio-EM lab for scientific support and discussions. The Scientific Computing Center at the University of Basel (sciCORE) and the National Supercomputing Centre Singapore (NSCC) are acknowledged for providing computational resources. This work was supported by the Swiss National Science Foundation (grants 167125, 185388 and 187170 to S.H. and R’Equip 177084 to T.M.), AntiResist: new approaches to combat antibiotic-resistant bacteria (51AU40_180541), the Medical Research Council (program grant MR/N020413/1 awarded to C.V.R.), the Bioinformatics Institute (BII) A*STAR and NRF (NRF2017NRF-CRP001-027 to P.J.B. and J.K.M.), and the NIH (grant P01 AI118687 to K.L.).
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C.V.R., K.L., T.M. and S.H. designed the study and supervised experiments. R.G. and Y.I. performed the microbiology experiments. J.R.B. performed the mass spectrometry experiments. H.K. and R.P.J. performed all other experiments. J.K.M. and P.J.B. ran simulations. E.A. performed sequence analysis. All authors analysed data and discussed the findings. H.K., K.L., T.M. and S.H. wrote the manuscript. All authors edited and approved the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Cryo-electron microscopy structure of the BAM–darobactin complex.
a, Flow chart of data processing to generate the structure (see Methods). b, Purified BAM–darobactin sample used for cryo-EM structure determination analysed on SDS–PAGE. This experiment was repeated at least three times independently with similar results. c, Representative electron micrograph of BAM–darobactin. This experiment was repeated at least three times independently with similar results. d, Selected examples of 2D classes from cryoSPARC. e, Viewing direction distribution plot for the final 3D reconstruction. f, Fourier shell correlation (FSC) curves for unmasked, spherical, loose and tight masks, and corrected FSC curve for the final reconstruction, yielding a gold standard FSC resolution of 3.03 Å. g, Local resolution variations of the EM reconstruction. POTRA domains 1 and 2 are at a local resolution below 4.5 Å and are visualized only at a lower contour level where micelle density obscures the view onto the BamA barrel. h, Plot of directional FSC (red; mean ± s.d.) and histogram of per angle FSC (blue). FSC curve indicates a resolution of 3.15 Å. i, Overview of the cryo-EM reconstruction of the BAM complex. BAM is shown in ribbon representation and the coulomb potential map as blue mesh. Note that the density of POTRA domains P1 and P2 is below the display threshold chosen here because of motional averaging. j–n, Expanded local views, showing the map around selected atoms in stick representation from the directions and viewpoints indicated by arrows and letters in i.
Extended Data Fig. 2 Structural details of the cryo-EM and crystal structures of BAM.
a, Superposition of the BAM–darobactin cryo-EM structure (salmon) with the ligand-free BAM crystal structure (green, PDB 5D0O). b, Superposition of POTRA domains P1–P5 and the individual components BamB–BamE, as indicated. The dashed horizontal line indicates the pivot between P2 and P3 around which P1 and P2 are rotated by rigid-body movement. c, Superposition of the BamA β-barrel–darobactin crystal structure (salmon) with a closed-gate BamA β-barrel crystal structure (cyan, PDB 4N75). d, Crystallographic omit map for darobactin bound at the lateral gate region of the BamA β-barrel after refinement of the model without darobactin. The 2mFo − DFc map is shown at 1σ in slate and the mFo − DFc difference map at ±3σ level in green and red. Top, overview of an entire BamA barrel. Bottom left and right, expanded views without and with overlay of the refined model coordinates, respectively. The cyclizations of darobactin can clearly be observed at 2.3 Å resolution. e, Omit map for strands β1 (top) and β16 (bottom) of the BamA β-barrel visualized as in d. f, Superposition of the cryo-EM (Bordeaux and white for BamA and darobactin, respectively) and X-ray structures (salmon and blue). g, As in f for the ligand darobactin only.
Extended Data Fig. 3 Comparison of BamA β-barrel conformations in aqueous solution.
a, Comparison of 2D [15N,1H]-TROSY fingerprint spectra of different BamA preparations in LDAO. Left, overlay of BamA-β fingerprint spectra in the absence and presence of darobactin. Middle, overlay of fingerprint spectra of BamA-β–nanobodyF7 and BamA-β–darobactin. Right, overlay of fingerprint spectra of BamA+9 (BamA-β + C-terminal extension MENVALDFS) and BamA-β–darobactin complex. Bottom panels show expanded views of boxed areas in main spectra. b, Backbone amide chemical shift perturbations between the fingerprint spectra of BamA-β with and without darobactin (left, black), Bam-β–nanobodyF7 in comparison with BamA–darobactin (middle, blue) and BamA+9 in comparison with BamA–darobactin (right, purple). The dotted lines indicate the average chemical shift perturbation (CSP), which can be interpreted as a measure of dissimilarity between two spectra. c, Top, structures of the BamA β-barrel in various conformations of the gate region. Bottom, expanded views of part of the backbone showing hydrogen bonds between β1 and β16 or darobactin. From left to right: open gate (6QGW, red); closed gate (6QGX, blue); BamA+9 (6FSU, purple); and BamA–darobactin complex. d, In vivo functional assay of BamA barrel mutants and C-terminal extensions using JCM166 cells in the absence and presence of arabinose. fl-BamAMENVALDFS and fl-BamA serve as a negative and positive controls, respectively.
Extended Data Fig. 4 ITC of BamA β-barrel in detergent micelles and its variants titrated with darobactin.
Experiments were repeated independently twice with similar results.
Extended Data Fig. 5 MD simulations of BamA β-barrel.
a, Representative snapshots of lipid PE molecule (left) and PG molecule (right) anchored by Ile430 and Leu780 in the gate region. b, The most dominant conformation of BamA–darobactin showing contacts consistently observed between the BamA β-barrel and darobactin throughout the simulation sampling. c, Partial densities of all lipids (top-down view of the membrane); the white arrow highlights the darobactin-binding region. d, Partial densities of lipid phosphate groups (side view of the membrane). e–g, Structural drift and fluctuations of key β-strands around the darobactin-binding site. Time-dependent r.m.s.d. measured with respect to the initial structure for backbone atoms of β-strands, after performing a least-squares fit. The resulting r.m.s.d. is shown for β16 in ligand-free BamA (e), β16 in BamA–darobactin complex (f), and hairpin β1/β2 in BamA–darobactin complex (g).
Extended Data Fig. 6 Interaction of BAM complex with lipids in the absence and presence of bound darobactin.
a, Mass spectra of the BAM complex with different lipids (top, CL; middle, PG; bottom, PE). b, Deconvolution of the mass spectra in a indicates that all the subcomplexes have lipids bound. c, Relative intensities of lipid binding peaks from a suggest that the negatively charged PG and CL have higher affinity than PE. d, Mass spectra of BAM complex with lipids and darobactin. Bottom, PE and darobactin; top, PG and darobactin. Below, expanded view of a section of the 23+ charge state highlights the bound peaks; bar charts show relative ratio of darobactin binding. No significant increase in darobactin binding is observed in these two cases, suggesting that PE and PG lipids do not affect darobactin binding. e, Mass spectra of BAM complex with lipid mixtures (bottom, PE and CL; top, PG and CL) and darobactin. Below, expanded view of a section of the 23+ charge state with lipids, darobactin and their various combination binding peaks highlighted. Bar charts of relative peak intensities indicate that darobactin bound with CL is observed to a greater extent than bound alone or bound with PE or PG. This increase is even higher for 2 × CL bound species and is slightly lower for PE or PG bound to 1 × CL species. However, no change in darobactin binding is observed for PE and PG. Bars (c–e) represent mean ± s.d., points show data from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant; two-tailed unpaired Student’s t-test. Exact P values are indicated in the figures.
Extended Data Fig. 7 MD simulations of the effect of darobactin-resistance mutations on the BamA-β–darobactin interaction.
a, Left, representative snapshots with expanded views from a simulation with strain 1 bound to darobactin. Mutations G429V and G807V are shown as yellow spheres at the α-carbon position. Right, time-dependent r.m.s.d. of β16 backbone atoms relative to the initial structure. b, As in a for strain 2 (mutations E435K and G443D, cyan) and r.m.s.d. of hairpin β1/β2. c, As in a for strain 3 (mutations T434A, Q445P and A705T, green) and r.m.s.d. of hairpin β1/β2. In each panel, protein is shown in cartoon representation, darobactin as sticks (blue, carbon; red, oxygen; white, proton; navy, nitrogen). Hydrogen bonds are shown as black dotted lines.
Extended Data Fig. 8 Relatedness of predicted Gram-negative BamA proteins.
a, Sequence alignment of BamA sequence from Gram-negative bacteria. The region highlighted in yellow is the predicted interaction site of darobactin in sheet β1. Right, these six-amino acid sequences from A. baumannii and B. fragilis were substituted into E. coli BamA for in vivo assays. b, Phylogenetic tree of full-length BamA sequences from various species of Gram-negative bacteria. Colours indicate branches belonging to the species specified next to the branch. Multiple alignments for the tree were carried out using CLUSTAL-W and the phylogenetic tree was derived using SEAVIEW software. c, Topology plot of BamA from E. coli with bound darobactin (blue). For the chimeric mutants, the red amino acids were exchanged with the local sequence from either A. baumannii or B. fragilis.
Extended Data Fig. 9 Comparison of BamA structures involved in molecular interactions and analysis of β-signals.
a, Overlay of the BamA subunit from the BAM–darobactin complex (salmon–blue; this work) with a BamA engaged with a substrate in a late-stage insertion intermediate state (green; PDB 6V05). The substrate has been omitted in this panel and the structures have been globally aligned to the protein backbone. b, Expanded view of the BamA β-barrel from a with strand β16subs of the substrate shown in purple. It is paired to strand β1mem of the catalytic BamA. Bold green and red arrows depict the directions of strand β1, forming an ~90° angle. c, Expanded view of the gate region indicating the spatial proximity of the substrate and darobactin interaction sites and their relative rotation of ~90°. d–f, Comparison of the register of β16 complementation to b1. d, In BamA–darobactin (salmon), residue Ile806 pairs with Tyr432. e, In the late-stage intermediate, Ile806 of the substrate BamAsubs (purple) pairs with Phe428 in catalyst BamAmem (green), corresponding to a register shift of 4. f, Hypothetical position of the four C-terminal residues of substrate BamA, which are not resolved in the available electron density. When paired to β1mem in canonical antiparallel β-strand conformation, they locate exactly at the darobactin-binding site, with the C-terminal Trp810 at the position of Phe7 of darobactin. g, Frequency logo of known and putative β-signals from bacterial OMPs, coloured by amino acid type. Numbering refers to distance from the C terminus. h, Distribution of log-likelihood scores in three sets of sequences, as indicated. The score obtained by the darobactin sequence is indicated by a blue line. The percentile rank of darobactin within each of the three sets is given in parentheses. i, j, As in g, h, but based on amino acid chemistry. H, hydrophobic and non-polar residue; A, aromatic; N, neutral; C, charged; P, polar non-charged.
Extended Data Fig. 10 Interaction of β-signal peptides with BamA β-barrel in detergent micelles by ITC.
The first four panels show direct titration of each of the ten-amino acid β-signal peptides of BamA, BtuB, FhuA, and OmpF to the BamA β-barrel. The next five panels show a competition experiment with darobactin titrated to the BamA β-barrel in the presence of ten-amino acid β-signal peptides of OmpT (0.7 mM), BtuB (2.6 mM), OmpF (1.4 mM) and FhuA (1.1 mM) and a β-consensus-peptide (1.2 mM). The results from fitting of the data to the competition model are given in Supplementary Table 7.
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Kaur, H., Jakob, R.P., Marzinek, J.K. et al. The antibiotic darobactin mimics a β-strand to inhibit outer membrane insertase. Nature 593, 125–129 (2021). https://doi.org/10.1038/s41586-021-03455-w
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DOI: https://doi.org/10.1038/s41586-021-03455-w
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