A new antibiotic traps lipopolysaccharide in its intermembrane transporter

No statistical methods were used to predetermine sample size. The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.

SDS–PAGE and immunoblotting

Homemade Tris-HCl 4–20% polyacrylamide gradient gels or 4–20% Mini-PROTEAN TGX precast protein gels (Bio-Rad) were used with Tris-glycine running buffer. The 2× SDS sample loading buffer refers to a mixture containing 125 mM Tris (pH 6.8), 4% (w/v) SDS, 30% (v/v) glycerol, 0.005% bromophenol blue and 5% (v/v) β-mercaptoethanol. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) gels were run for 45–60 min at 200 V. Protein complexes purified for cryo-EM were analysed by SDS–PAGE followed by staining with Coomassie blue (Alfa Aesar) and imaging using the Gel feature of an Azure Biosystems C400 imager. For western blotting, proteins were transferred onto Immun-Blot PVDF membranes (Bio-Rad). Membranes were then blocked using sterile-filtered Casein blocking buffer (Sigma-Aldrich) for 1 h and subsequently incubated with the appropriate antibodies. The following primary antibodies were used: mouse anti-His HRP conjugate (Biolegend, 652504, 1:10,000 dilution) and anti-LPS core mouse monoclonal (Hycult Biotechnology, HM6011, clone WN1 222-5, lot no. 18419M0715-A, 1:5,000 dilution). The following secondary antibodies were used: donkey-anti-rabbit RP conjugate (GE Amersham, NA934-1ML, lot no. 16801031, 1:10,000 dilution), sheep-anti-mouse HRP conjugate (GE AMersham, LNA931V/AH, lot no. 14251045, 1:10,000 dilution). Bands were visualized using ECL Prime western blotting detection reagent (GE Amersham) and an Azure c400 imaging system. Uncropped immunoblots are available in Supplementary Fig. 1.

Plasmids, strains and oligonucleotides

Genes encoding the LptB, LptC and LptFG were amplified by polymerase chain reaction (PCR) from Acinetobacter baylyi ADP1 (ATCC 33305) genomic DNA. lptB and lptFG PCR products were inserted into pCDFduet by Gibson assembly (New England Biolabs) to generate plasmids analogous to those used for other LptB2FG homologues16. Similar design was used for the modified plasmid pTRAB-FLAG-LptB-LptFG for purification of the same complex from the native host, which was constructed by combining the gDNA amplicons of the same open reading frames incorporating a linker-less FLAG tag at the N terminus of LptB, a modified trp promoter of E. coli and adjacent regions from pTRC99a, a hybrid pBR322WH1266 replicon and a spectinomycin resistance cassette from pCDFduet by Gibson assembly. A linker-less N-terminal His7 tag was added to LptB in pCDFduet using NEBuilder HiFi DNA assembly (New England Biolabs). lptC PCR products were inserted into pET22/42 with a C-terminal thrombin cleavage site and a His7 tag. Oligonucleotide primers were purchased from Eton Biosciences, Genewiz or Integrated DNA Technologies. Plasmids and strains used in this study are reported in Supplementary Tables 4 and 5, respectively. Plasmid sequences are below.

Construction and use of mutant A. baylyi strains

Culture, genetic manipulation and MIC measurements of A. baylyi ADP1 were conducted according to previously reported procedures43,48. Point mutants were constructed in a two-step procedure following ref. 49 with the introduction and excision of the integration cassette at codon 66 of pepA, wherein the excising fragment of otherwise wild-type chromosomal DNA sequence from codon 406 of pepA to codon 193 of lptG bore the desired mutation and the resulting clones were screened by amplicon sequencing from codon 81 of HolC to codon 501 of GpmI, whereas lpxM deletion was achieved following the same procedure except that the integration cassette insertion and excision removed codons 79–279 of lpxM to avoid interference with neighbouring and overlapping genes that a larger deletion may risk and replaced codon 78 with an ochre stop codon to prevent a readthrough resulting in an aberrant fusion, with the excising fragment sequence spanning from codon 211 of sppA to codon 497 of ComA and verified by amplicon sequencing from codon 94 of MhpC to codon 327 of ComA as well as by absence of a PCR product corresponding to a region spanning codons 79 to 279 of lpxM to check for duplications. A deletion of the lon protease was made in the same manner to produce the stain used for expression and purification of LptB2FG to mimic the BL21 strain of E. coli used in the rest of purifications, for which the region encompassing 72 base pairs (bp) upstream of the lon start codon and 1 bp downstream of the lon stop codon was excised after being replaced with the same integration cassette, yielding a markerless deletion, with the excising fragment sequence spanning from codon 491 of ArnT to codon 40 of 45_DOPA_Dioxygenase and verified by amplicon sequencing spanning from codon 322 of ArnT to codon 221 of 45_DOPA_Dioxygenase as well as by absence of a PCR product corresponding to a region spanning from codons 328 to 768 of lon to check for duplications. Following amplicon confirmation, three validated isolates of each constructed mutant were tested for susceptibility to a panel of antibiotics with known antibiotics with known mechanisms of action as a further validation step to ensure congruence of phenotypes across replicates, which was confirmed in all cases and one of the validated replicates was later used for MIC measurements reported here. In the case of R30A and R55G, no colonies that incorporated these mutations could be isolated, whereas the identical approach readily introduced conservative R30K and R55K substitutions, which resulted in increased antibiotic sensitivity in spite of their mild nature, indicating that impairments caused by substituting dissimilar residues at those positions are not survived.

MIC determination

MIC determinations were performed by broth microdilution in line with CLSI guidelines (CLSI M07-A11 2018). Bacterial inocula were prepared by diluting overnight liquid cultures in LB. Antibacterial panels containing antibacterial solutions were inoculated with an appropriate volume of inoculum to give a final inoculum of about 5 × 105 c.f.u. ml−1 and desired test concentrations of antibacterial agents in standard 96-well plates with 0.1 ml of culture per well. The test plates were incubated for 20–24 h and optical density (OD600) was recorded using a plate reader. MIC values corresponded to the lowest compound concentration inhibiting bacterial growth beyond which OD ceased to decrease.

Purification of LptB2FG complexes for cryo-EM

LptB2FG complexes were purified as previously described, with slight modifications17. Overnight cultures of Bl21(λDE3) E. coli containing pCDFduet-His7LptB-LptFG or A. baylyi containing pTRAB-FLAGLptB-LptFG were diluted 1:100 into LB or terrific broth containing 50 mg l−1 of spectinomycin. Cells were grown at 37 °C (or 30 °C for A. baylyi) to an OD600 of about 0.8. Then 200 µM IPTG and 0.2% glucose (or 500 µM IPTG for A. baylyi) were added and cells were allowed to grow for another 2–3 h. Cells were harvested by centrifugation (4,200g, 20 min, 4 °C). Cell pellets were flash frozen using liquid nitrogen and stored at −80 °C. All subsequent steps were carried out at 4 °C unless otherwise noted.

Thawed cell pellets were resuspended in lysis buffer (50 mM Tris (pH 7.4), 300 mM NaCl, 1 mM PMSF, 100 μg ml−1 of lysozyme, 50 μg ml−1 of DNase I, 1 cOmplete Protease Inhibitor Cocktail tablet per 40 ml) homogenized and subjected to passage through an EmulsiFlex-C3 high-pressure cell disruptor three times. The cell lysate was centrifuged (10,000g, 10 min) and the supernatant was further centrifuged (100,000g, 1 h). The resulting pellets were resuspended and solubilized in solubilization buffer (20 mM Tris (pH 7.4), 300 mM NaCl, 15% glycerol, 5 mM MgCl2, 1% (wt/vol) DDM (Anatrace Maumee), 100 μM PMSF, 2 mM ATP) and rocked at 4 °C for 2 h. (A. baylyi cell lysate was immediately subjected to detergent solubilization without the preceding centrifugation steps or ATP addition but 0.35 µM 1 was used to supplement some batches from the solubilization step onward). The mixture was centrifuged (100,000g, 30 min), the supernatant was spiked with imidazole to a final concentration of 15 mM and then rocked with Ni-NTA Superflow resin (Qiagen) for 1 h. (A. baylyi supernatant was also filtered through a 0.45 µM pore size PVDF Durapore (Millipore-Sigma) membrane and incubated with M2-FLAG agarose resin (Millipore-Sigma) without imidazole supplementation instead of Ni-NTA Superflow resin). The resin was then washed with 2 × 10 column volumes affinity buffer (300 mM NaCl, 20 mM Tris (pH 7.4), 15% glycerol, 0.01% (wt/vol) DDM, 0.04% (wt/vol) GDN (Anatrace Maumee)) containing 20 mM imidazole followed by 2 × 15 column volumes of affinity buffer containing 35 mM imidazole. (A. baylyi-derived batches were washed with 3 × 10 column volumes of affinity buffer). Protein was eluted with 2 × 2 column volumes of affinity buffer containing 200 mM imidazole (12.5 column volumes of affinity buffer supplemented with 0.2 mg ml1 of FLAG peptide (Genscript) for A. baylyi-derived batches) concentrated using a 100 kDa molecular weight cutoff Amicon Ultra centrifugal filter (Millipore) and purified by size-exclusion chromatography on a Superdex 200 increase column in SEC buffer (300 mM NaCl, 20 mM Tris (pH 7.4), 0.02% GDN, 0.25 mM tris(hydroxypropyl)phosphine). Fractions were pooled and concentrated to 7–8 mg ml−1 using a 100 kDa molecular weight cutoff Amicon Ultra centrifugal filter. Protein was then prepared for microscopy as described below.

Purification of LptB2FGC complexes for cryo-EM

Purification was conducted largely as described for LptB2FG with the following modifications. Expression was conducted using C43(λDE3) E. coli containing pCDFduet-LptB-LptFG and pET22/42-LptC-thrombin-His7. Cultures were grown in the presence of 50 mg l−1 of spectinomycin and 50 mg l−1 of carbenicillin. The rest of the expression and purification was conducted identically to the LptB2FG purification until the size-exclusion chromatography step. Fractions collected after size-exclusion chromatography were incubated overnight with restriction-grade thrombin (Sigma) to cleave the His tag. The solution was spiked with 8 mM imidazole and the uncleaved protein was removed by passage through Ni-NTA resin and benzamidine Sepharose. Fractions were pooled and concentrated to 7–8 mg ml−1 using a 100 kDa molecular weight cutoff Amicon Ultra centrifugal filter. Protein was then prepared for microscopy as described below.

Electron microscopy data collection

Protein was purified as described above and then incubated on ice with 0.2 mg ml−1 of lipopolysaccharides from E. coli EH100 (Ra mutant; Sigma-Aldrich) and 0.25 mM drug (if applicable) for 45 min with gentle agitation. For proteins purified out of Acinetobacter, E. coli lipopolysaccharides were not added. Sample was then applied to glow-discharged C-flat 20 nm holey carbon 1.2 μm hole diameter, 1.3 μm hole spacing, 400-mesh copper grids (Protochips). Grids were blotted for 6.5 s at 4 °C and 100% humidity with the blot force set to 12 and flash frozen by liquid nitrogen-cooled liquid ethane using a Thermo Fisher Scientific Vitrobot Mark IV (Thermo Fisher Scientific). The grid was then loaded onto a Titan Krios G3i electron cryo-microscope (Thermo Fisher) operated at 300 kV accelerating voltage. Image stacks (videos) were recorded on a Gatan Bioquantum K3 Imaging Filter (Gatan), using counting mode and a calibrated magnification of ×105,000 and a pixel size of 0.825 Å, using SerialEM50. The slit of the energy filter was set to 20 eV with a defocus range between 1.1 and 2.2 μm. The subframe time was set to allow the collection of 50 subframes per image stack with an electron dose rate of about 1 e per Å2 per frame. The total electron dose was about 50 e per Å2. The multishot scheme in SerialEM was used for data collection, with settings of nine holes per stage move and two shots per hole. The data collections for all structures were performed in the same manner.

Image processing and three-dimensional reconstruction

The video frames were motion-corrected and dose-weighted and the contrast transfer function (CTF) parameters were estimated using CryoSPARC Live51. Particle picking was carried out using the cryoSPARC blob-picker and junk particles were filtered out by successive rounds of two-dimensional classification in cryoSPARC. Initial models were generated using the ab initio reconstruction in cryoSPARC and then particles were filtered by successive rounds of heterogeneous refinement. After an initial non-uniform refinement job, particles were subject to local motion correction, patch CTF estimation, local CTF refinement and global CTF refinement (fit for beam tilt, beam trefoil and spherical aberration). The particles were then subject to non-uniform refinement to yield the final global reconstruction. Maps were further refined using particle subtraction and local refinement with a mask focused on the TM and nucleotide-binding domains of the transporter. For all maps, we also tried classification without alignment in Relion. At best this only yielded nominal improvements in resolution after reimporting into cryoSPARC and conducting non-uniform refinement when compared to the preclassification maps. 3D classification without alignments in cryoSPARC revealed several possible conformations of the drug within the transporter, as highlighted in Extended Data Fig. 3b,f52,53. Maps used for figures were either filtered according to local resolution with B-factor sharpening within cryoSPARC or using postprocessing carried out in DeepEMhancer54. Structural biology applications used in this project were compiled and configured by SBGrid55.

Model building, refinement and validation

Initial models for LptB, LptF and LptG were generated using SwissModel56. The resulting structures were docked into the LptBFG map using Chimera57. Cif restraints for E. coli lipopolysaccharide were generated using the sketcher tool in CCP4 (ref. 58). Cif restraints for Acinetobacter lipopolysaccharide were generated using the Grade2 web server from Global Phasing Limited. Cif restraints for the macrocyclic peptides were generated using eLBOW59. The coordinates were then refined using Phenix60,61. The model was further optimized using ISOLDE62, accessed through ChimeraX63. Manual model building was carried out in Coot64. The final model was visually inspected for general fit to the map and further inspected using MolProbity and the residue-wise local quality estimation DAQ65,66. All residues in our models had >0 DAQ scores, except those contained in the helix of LptC. The helix of LptC is modelled as poly-alanine because our maps were not of sufficient quality to allow unambiguous assignment of the helix register. The model validation statistics are summarized in Extended Data Table 1.

Purification of LptB2FG complexes for biochemical reconstitution

LptB2FG used for biochemical experiments was purified as described for cryo-EM with the following modifications. The affinity buffer was 300 mM NaCl, 20 mM Tris (pH 7.4), 10% glycerol, 0.015% (wt/vol) DDM. The SEC buffer was 300 mM NaCl, 20 mM Tris (pH 7.4), 5% glycerol, 0.05% DDM, 0.5 mM tris(hydroxypropyl)phosphine.

Purification of LptB2FGC complexes for biochemical reconstitution

LptB2FGC used for biochemical experiments was purified as described for cryo-EM with the following modifications. The affinity buffer was 300 mM NaCl, 20 mM Tris (pH 7.4), 10% glycerol, 0.015% (wt/vol) DDM. The SEC buffer was 300 mM NaCl, 20 mM Tris (pH 7.4), 5% glycerol, 0.05% DDM, 0.5 mM tris(hydroxypropyl)phosphine. E. coli LptB2FGC complexes were purified as described previously17.

Purification of LptAI36pBPA

LptAI36pBPA was purified as described previously17. Briefly, Bl21 (λDE3) E. coli cells containing pSup-BpaRS-6TRN and pET22b-LptA(I36Am) were grown to an OD600 of approximately 0.6 at 37 °C in LB media containing 50 μg ml−1 of carbenicillin, 30 μg ml−1 of chloramphenicol and 0.8 mM pBPA (BaChem). Cells were then induced with 50 μM IPTG; allowed to grow for 2 h; harvested; resuspended in a mixture containing 50 mM Tris-HCl (pH 7.4), 250 mM sucrose and 3 mM EDTA; incubated on ice for 30 min; and pelleted (6,000g, 10 min). The supernatant was supplemented with 1 mM PMSF and 10 mM imidazole and pelleted (100,000g, 30 min). The supernatant was incubated with Ni-NTA resin, which was then washed twice (20 column volumes of 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% (vol/vol) glycerol and 20 mM imidazole). LptA was eluted twice (2.5 column volumes of wash buffer supplemented with a further 180 mM imidazole), concentrated using a 10-kDa-cutoff Amicon centrifugal concentrator (Millipore), flash frozen and stored at −80 °C until use.

Preparation of LptB2FG or LptB2FGC liposomes

Proteoliposomes were prepared as described previously17. Aqueous E. coli polar lipid extract (Avanti Polar Lipids) (30 mg ml−1) and aqueous LPS from E. coli EH100 (Ra mutant; Sigma) (2 mg ml−1) were sonicated briefly for homogenization. For experiments testing the effect of LPS structure, we used LPS isolated from either GKM374 (BL21DE3 eptA::catR arnA::kanR eptC::gentR) or TXM418 (BL21DE3 eptA::catR arnA::FRT eptB::gentR lpxM::kanR) as described previously33. A mixture of 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 7.5 mg ml−1 of E. coli polar lipids, 0.5 mg ml−1 of LPS and 0.25% DDM was prepared and kept on ice for 10 min. Purified LptB2FGC or LptB2FG was added to a final concentration of 0.86 μM and the mixture was left on ice for 20 min. The mixture was diluted 100-fold with cold 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl and kept on ice for 20 min. The proteoliposomes were pelleted (300,000g, 2 h, 4 °C), resuspended in 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl, diluted 100× and centrifuged (300,000g, 2 h, 4 °C). The pellets were resuspended in a mixture of 20 mM Tris-HCl (pH 8.0), 150 mM NaCl and 10% glycerol (250 μl per 100 μl of the original predilution solution), homogenized by sonication, flash frozen and stored at −80 °C until use.

Purification of LptC(ΔTM)

LptC(ΔTM) was purified largely as previously described16. Briefly, Bl21 (λDE3) E. coli cells containing pET22/42-LptC(ΔTM)-His7 were grown to an OD600 of approximately 0.6 at 37 °C in LB media containing 50 μg ml−1 of carbenicillin. Cells were then induced with 50 μM IPTG; allowed to grow for 2 h; harvested and resuspended in lysis buffer (50 mM Tris pH 7.4, 300 mM NaCl, 0.1 mM EDTA). Lysozyme, DNaseI and PMSF were added to final concentrations of 100 µg ml−1, 50 µg ml−1 and 1 mM, respectively. Cells were homogenized and subjected to passage through an EmulsiFlex-C3 high-pressure cell disruptor three times. The cell lysate was centrifuged (10,000g, 10 min) and the supernatant was further centrifuged (100,000g, 1 h). The supernatant was spiked with imidazole to a final concentration of 15 mM and then rocked with Ni-NTA Superflow resin (Qiagen) for 1 h. The resin was then washed with 2 × 10 column volumes of affinity buffer (300 mM NaCl, 20 mM Tris (pH 7.4), 15% glycerol) containing 20 mM imidazole followed by 2 × 15 column volumes of affinity buffer containing 35 mM imidazole. Protein was eluted with 2 × 2 column volumes of affinity buffer containing 200 mM imidazole, concentrated using a 10 kDa molecular weight cutoff Amicon Ultra centrifugal filter (Millipore) and purified by size-exclusion chromatography on a Superdex 200 increase column in SEC buffer (300 mM NaCl, 20 mM Tris (pH 7.4), 5% glycerol). Fractions were pooled and stored at −80 °C.

LPS release assay

The amounts of release of LPS from proteoliposomes to LptA were measured as previously described17. Assays used 60% proteoliposomes (by volume) in a solution containing 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10% glycerol and 2 µM LptAI36pBPA. Reaction mixtures were incubated with drug for 10 min at room temperature, as applicable. Reactions were then initiated by the addition of ATP and MgCl2 (final concentrations of 5 mM and 2 mM, respectively) and proceeded at 30 °C. Aliquots (25 µl) were removed from the reaction mixtures and irradiated with ultraviolet (UV) light (365 nm) on ice for 10 min using a B-100AP lamp (Fisher Scientific). Following UV irradiation, 25 µl of 2× SDS–PAGE sample loading buffer was added, samples were boiled for 10 min and proteins were separated using Tris-HCl 4–20% polyacrylamide gradient gels with Tris-glycine running buffer. Immunoblotting was conducted as described above.

ATPase assay

ATPase assays were done using a modified molybdate method, as previously reported, with slight modifications17. Assays used 30% proteoliposomes (by volume) in a mixture containing 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10% glycerol and 2 mM MgCl2. Proteoliposome-containing reaction mixture was incubated with drug at room temperature for 10 min, as applicable. Reactions were initiated by the addition of ATP to a final concentration of 5 mM and run at 30 °C. Aliquots (5 µl) were taken at 0, 15, 30 and 45 min. Reactions were quenched with an equal volume of 12% SDS. The amounts of Pi were determined using a colorimetric method and potassium phosphate was used as a standard43. Reagents were obtained from Sigma-Aldrich. After the addition of SDS, a mixture containing 10 µl of 30 mg ml−1 of ascorbic acid, 0.5 N HCl, 5 mg ml−1 of ammonium molybdate and 6% SDS was added. The samples were incubated at room temperature for 7 min and 15 µl of an aqueous solution containing 20 mg ml−1 of sodium citrate tribasic dihydrate, 2 mg ml1 of sodium arsenite and 2% (vol/vol) acetic acid was added. The absorbance at 850 nm was measured using a Spectramax Plus 384 (Molecular Devices) after 20 min. Error bars indicate the standard deviations of the average rates measured over three biological replicates.

Scintillation-proximity assay

The binding of radioligand [3H]-RO7223280 to ablLptB2FG and ablLptB2FGC was measured by bead-based SPA. All steps were performed in SEC buffer (20 mM Tris pH 7.5, 300 mM NaCl, 5% glycerol, 0.5 mM TCEP, 0.05% DDM ± 10 µM E.coli J5 LPS(Rc) TLRGRADE (Enzo Life Sciences)) and at 4 °C unless otherwise indicated. Purified protein was first incubated with Copper PVT HIS-tag beads (Perkin Elmer) for 1.5 h under gentle rotation. Twelve radioligand concentrations were added to the PVT–protein mix and incubated for another 30 min. The mixture was diluted into SPA buffer without or with 500 nM cold RO7223280 to measure total and non-specific binding respectively into an Optiplate-384 microplate (Perkin Elmer). Each well contained 25 ul of total volume, 18 nM protein, 5% dimethylsulfoxide and 6% v/v PVT beads. The SPA plates were sealed (TopSeal, Perkin Elmer) and stored at 4 °C overnight. Before the measurement, plates were mixed on a shaker for 20 min, 750 rpm at room temperature and the seal was thoroughly wiped with antistatic spray to reduce electrostatic events. Scintillation data were recorded with a Topcount NXT C384, in the form of three independent replicates each consisting of three technical triplicates. Specific binding was calculated by subtracting non-specific binding raw counts from total binding raw counts. The dissociation constant Kd and standard deviation of the three independent replicates are reported and were calculated by the GraphPad Prism ‘One site – specific binding’ tool.

Radioligand displacement experiments were similarly conducted, applying a constant concentration of 25 nM [3H]-RO7223280 and 16 concentrations of cold ligands in the presence of 10 µM LPS(Rc). Displacement values were normalized by including nine wells containing no radioligand (defined as 100% competition) and nine wells containing 8 uM radioligand only (defined as 0% competition). The inhibitory constant Ki was calculated with the ‘One Site – Fit Ki‘ tool using the concentration (25 nM) and Kd (86 nM) of [3H]-RO7223280 as constraints. The Hill coefficient was used as a quality control metric (theoretically, nH = 1 for a 1:1 competitive inhibitor) and determined with the ‘[Inhibitor] vs. response — Variable slope (four parameters)’ tool.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.


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