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Structural basis for inhibition of the drug efflux pump NorA from Staphylococcus aureus

Nature Chemical Biology volume 18, pages 706–712 (2022)Cite this article

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Abstract

Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA–Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate-binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with submicromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.

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Fig. 1: NorA–Fab complex structures determined using single-particle cryo-EM.
Fig. 2: Identification of key residues within NorA essential for drug resistance.
Fig. 3: Fab binding and inhibition of NorA.
Fig. 4: NorA binding and MRSA growth inhibition by a peptide mimicking CDRH3.

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Data availability

The datasets generated during and/or analyzed during the current study are deposited in the Electron Microscopy Data Bank and PDB for NorA–Fab25 (EMD-23463; PDB ID: 7LO7) and NorA–Fab36 (EMD-23464; PDB ID: 7LO8). Source data are provided with this paper.

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Acknowledgements

This work was supported by NIH (R01AI165782, R01AI108889) and NSF (MCB 1902449) awards to N.J.T., NIH awards (R01AI165782, R01NS108151, R01GM121994, R01DK099023) to D.-N.W., NIH award (R01AI165782) to S.K., NIH awards (R01AI099394, R01AI105129, R01AI137336, R01AI140754, R21AI149350) to V.J.T. and an NIH award (R35GM130333) to P.S.A. V.J.T. is a Burroughs Wellcome Fund Investigator in the pathogenesis of infectious diseases. D.N.B. was supported by an NIH Predoctoral Training Grant (T32-GM088118). D.B.S. was supported by an American Cancer Society Postdoctoral Fellowship (129844-PF-17-135-01-TBE) and a Department of Defense Horizon Award (W81XWH-16-1-0153). We thank D. C. Hooper for sharing a NorA construct in the pTrcHis2C vector and J. F. Hunt for the MSP1E3D1-T277C construct. We thank S. Wang for assistance in optimizing computational resources for cryo-EM data processing at the NYU HPC, M. (Leninger) Crames and A. Tumati for initial NorA experiments that identified LMNG as a suitable solubilizing detergent, P. Tate for assistance in cloning and I. Irnov, N. K. Karpowich, J. J. Marden and W. J. Rice for helpful discussions. Negative stain and cryo-EM grids were screened at the Microscopy Facility and the Cryo-Electron Microscopy Facility of the NYU School of Medicine, respectively. We thank the Pacific Northwest Cryo-EM Center (PNCC) and laboratory personnel who assisted in the collection of the large cryo-EM datasets used for structure determination. EM data processing used computing resources at the HPC facilities of NYU and NYU School of Medicine. The following reagent was provided by the Network on Antimicrobial Resistance in S. aureus (NARSA) for distribution by BEI Resources, NIAID, NIH: S. aureus subsp. aureus, strain JE2, transposon mutant NE1034 (SAUSA300_0680), NR-47577. The pBAD33-Gm vector was obtained through Addgene (plasmid 65098; http://n2t.net/addgene:65098; RRID:Addgene_65098).

Author information

Author notes

  1. David B. Sauer

    Present address: Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, UK

  2. Zheng Liu

    Present address: Kobilka Institute of Innovative Drug Discovery, School of Life and Health Sciences, The Chinese University of Hong Kong, Shenzhen, China

  3. These authors contributed equally: David B. Sauer, Jianping Li, Xuhui Zheng, Akiko Koide.

Authors and Affiliations

  1. Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY, USA

    Douglas N. Brawley, David B. Sauer, Jinmei Song & Da-Neng Wang

  2. Department of Chemistry, New York University, New York, NY, USA

    Jianping Li, Ganesh S. Jedhe, Tiffany Suwatthee, Paramjit S. Arora & Nathaniel J. Traaseth

  3. Department of Microbiology, New York University School of Medicine, New York, NY, USA

    Xuhui Zheng & Victor J. Torres

  4. Perlmutter Cancer Center, New York University School of Medicine, New York, NY, USA

    Akiko Koide & Shohei Koide

  5. Department of Medicine, New York University School of Medicine, New York, NY, USA

    Akiko Koide

  6. Cryo-Electron Microscopy Facility, New York University School of Medicine, New York, NY, USA

    Zheng Liu

  7. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA

    Shohei Koide

  8. Antimicrobial-Resistant Pathogens Program, New York University School of Medicine, New York, NY, USA

    Victor J. Torres

  9. Department of Cell Biology, New York University School of Medicine, New York, NY, USA

    Da-Neng Wang

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  1. Douglas N. Brawley
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Contributions

D.N.B. optimized NorA expression and purification protocols, screened Fab fragments, prepared samples for cryo-EM, performed binding assays, troubleshot cryoSPARC installation, processed and analyzed cryo-EM datasets, built atomic models, interpreted the structures, performed resistance assays of NorA mutations in E. coli, was involved in project design and contributed to writing the manuscript. D.B.S. performed negative stain electron microscopy, froze grids for cryo-EM, processed and analyzed cryo-EM datasets, built atomic models and interpreted the structures. J.L. performed the NorA–Fab inhibition experiment in E. coli, performed binding assays, carried out immunoblotting analyses and prepared peptide stocks for MRSA inhibition experiments. X.Z. and V.J.T. designed and performed genetics, MIC and growth inhibition experiments in MRSA and prepared membrane fractions from MRSA for immunoblotting analyses. A.K. and S.K. screened and identified Fab clones that bind to NorA. G.S.J. and P.S.A. designed and generated peptides mimicking CDRH3. T.S. performed FP experiments with FITC–NPI-1 and the norfloxacin competition experiment. J.S. purified the membrane scaffold protein and performed NorA nanodisc reconstitution for Fab generation. Z.L. collected preliminary cryo-EM datasets. D.-N.W. directed and designed the project and contributed to writing the manuscript. N.J.T. directed and designed the project and wrote the manuscript. All authors participated in data analysis and in revising the manuscript.

Corresponding authors

Correspondence to Shohei Koide, Da-Neng Wang or Nathaniel J. Traaseth.

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Competing interests

S.K. is a SAB member and holds equity in and receives consulting fees from Black Diamond Therapeutics, a co-founder of and holds equity in Revalia Bio, and receives research funding from Puretech Health and Argenx BVBA. V.J.T. is an inventor on patents and patent applications filed by NYU, which are currently under commercial license to Janssen Biotech Inc. Janssen Biotech Inc. provides research funding and other payments associated with the licensing agreement. All other authors declare no competing interests. NYU has filed a provisional patent application covering the NorA inhibitors described in this work.

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Nature Chemical Biology thanks Aravind Penmatsa, Zhao Wang 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 Preparation of NorA samples for Fab screening and cryo-EM.

a. Purification of NorA in LMNG detergent. Left: SEC chromatogram displaying absorbance at 280 nm (Abs280nm) for NorA in LMNG. Right: Coomassie-stained SDS-PAGE gel of peak fractions from the SEC chromatogram. Similar results were obtained in ~15 independent experiments. b. Coomassie-stained SDS-PAGE gel showing successful reconstitution of NorA (His-tagged) into biotinylated membrane scaffold protein (MSP) nanodiscs for phage display screening of Fabs. The sample was passed over a Ni-NTA column with the imidazole elution displaying both NorA and MSP bands. Similar reconstitution results were obtained in three independent experiments. c. SEC chromatograms of NorA in PMAL-C8 amphipol alone (black traces) and NorA in the presence of three-fold molar excess of Fab25 (top, magenta) or Fab36 (bottom, blue). Major fractions from the left-shifted peaks indicated by arrows were collected and applied onto cryo-EM grids for structure determination. d, e. MST binding curves of fluorescently labelled Fabs to NorA reconstituted in PMAL-C8 amphipol. Kd values are shown next to each dataset; ‘n.d.’ refers to not determined. The error in Kd values for Fab25 (1.2 ± 0.7 μM) and Fab36 (140 ± 20 nM) represent the average and standard deviation of two independent runs with three or four replicates per independent run. The error range in the Kd value for Fab36W133A (2 to 4 μM) corresponds to the 95% confidence interval of the non-linear fit. The Fab36R134A binding curve could not be accurately fit and the Kd value was estimated to be greater than the highest concentration of NorA in the experiment (> 14 μM). MST binding experiments with Fab36W133A and Fab36R134A were collected with three replicates each.

Source data

Extended Data Fig. 2 Workflow for NorA-Fab36 complex structure determination by cryo-EM.

Key steps in cryo-EM data processing are displayed for determination of the coulomb potential map of the NorA-Fab36 complex. Solid and dotted boxes around classes correspond to the intact NorA-Fab36 complex and a NorA-Fab36 complex apparently lacking the constant Fab region, respectively. Underneath each class is the number of particles constituting that class. The percentages in the figure indicate the percentage of particles selected for the subsequent refinement step from the total number of particles initially inputted into the refinement step. An identical strategy was used for processing images of the NorA-Fab25 complex.

Extended Data Fig. 3 Cryo-EM structure determination of NorA-Fab25 and NorA-Fab36 complexes.

a, b. Representative cryo-EM micrographs (top) and exemplary 2D class averages (bottom) for NorA-Fab25 (a) and NorA-Fab36 (b) complexes. Images were acquired on a 300 kV Krios microscope. The most populous 2D classes show intact NorA-Fab complexes in different orientations. c, d. Fourier shell correlation (top) and directional FSC (bottom) curves measured for the final NorA-Fab25 (c) and NorA-Fab36 (d) reconstructions. The gold standard resolution (FSC = 0.143) is indicated by an arrow. Histograms within the directional FSC plots (bottom) correspond to the y-axis on the right. e, f. Orientation distribution heatmaps for the final NorA-Fab25 (e) and NorA-Fab36 (f) reconstructions. g, h. Coulomb potential maps (left) and local resolution maps (right) of NorA-Fab25 (g) and NorA-Fab36 (h). The local resolution maps are illustrated with the same linear coloring scale, where blue corresponds to regions of higher resolution and red corresponds to regions of lower resolution.

Extended Data Fig. 4 Quality assessment of model-to-map fitting for the NorA-Fab complexes.

a, b. The quality of the NorA-Fab25 (a) and NorA-Fab36 (b) models are illustrated by their respective fit into the experimental maps (shown in mesh). The model-to-map fitting is shown for each TM helix in NorA and a portion of the CDRH3 loop from each Fab. The map contour level was set to 10 sigma units for each TM helix and CDRH3 loop using the isomesh command in PyMOL. The following residues comprise each TM domain: TM1, 2-26; TM2, 37-63; TM3, 69-87; TM4, 91-115; TM5, 125-148; TM6, 155-175; TM7, 203-228; TM8, 238-264; TM9, 270-287; TM10, 297-320; TM11, 325-351; TM12, 356-374.

Extended Data Fig. 5 NorA topology and structure.

a. Left: Structure of NorA from the NorA-Fab36 complex where the colors correspond to the 12-TM helices of NorA, as depicted in the cartoon atop the structure. Right: Superimposition of NorA from the NorA-Fab25 structure (blue) with NorA from the NorA-Fab36 structure (red). The all-atom RMSD is 1.2 Å and the Cα RMSD is 0.9 Å. b. Structural views of NorA from the NorA-Fab36 complex. Left: Sequence alignment of NorA with the consensus sequences for Motif A and Motif C in MFS proteins8,26. Identical residues are shaded in grey and lowercase residue codes correspond to less conserved sites than the uppercase residue codes. Middle: Structure of NorA with insets showing expanded views from of Motif A and all of Motif C. The distance (in Å) between the carboxyl side chain of Asp63 (TM2) and the backbone amide of Arg324 (TM11) is shown with a yellow dotted line. Middle, Right: TM4 (cyan), TM5 (green), TM10 (orange), and TM11 (red) seal the substrate-binding pocket by forming interhelical contacts on the cytoplasmic side of the membrane. Phe129 from TM5 helps seal the substrate pocket by forming a plug in the middle of these TM helices. c. Cartoon representation of NorA from the NorA-Fab36 structure displaying the four ionizable residues (green sticks) within the substrate-binding pocket: Arg98 (TM4), Glu222 (TM7), Asp307 (TM10), and Arg310 (TM10).

Extended Data Fig. 6 NorA-Fab binding interfaces.

a, b. Detailed views of CDRH3 interactions in the substrate-binding pocket of NorA (rainbow) for Fab25 (panel (a); pink) and Fab36 (panel (b); blue). The cryo-EM maps for CDRH3 and select residues of NorA are superimposed on the structural views (grey mesh). c. Left: NorA (grey) interacting with CDRH3 from Fab25 (top; magenta) and Fab36 (bottom; blue). NorA residues within 4 Å of CDRH3 residues are labeled and displayed in a stick representation (orange). TM2 and TM11 within NorA are hidden for clarity. Right: View from the outward-facing direction and orthogonal to the membrane surface. NorA (grey) is shown in a surface representation with the magenta and blue colors indicating NorA residues within 4 Å of Fab25 CDRH3 or Fab36 CDRH3, respectively. Fab25 and Fab36 are not shown for clarity. d. Left: NorA (grey) interacting with an elongated loop of Fab36 with select residues displayed in stick representation (green). This loop is formed by scaffold residues Leu71 to Arg85 of the Fab light chain variable domain. The CDRH3 loop is shown in ribbon (light blue). TM2 and TM11 within NorA are hidden for clarity. Right: View from the outward-facing direction and orthogonal to the membrane surface. NorA (grey) is shown in a surface representation with the green color indicating NorA residues within 4 Å of the Fab36 light chain scaffold loop. Fab36 is not shown for clarity.

Extended Data Fig. 7 Expression and function of NorA mutants in MRSA and E. coli.

a. Dilution experiment in MRSA and MRSAΔnorA strains to test bacteriostatic or bactericidal effect of norfloxacin. Cells were grown overnight to saturation, diluted 1,000-fold into fresh media containing norfloxacin at 0, 12.5, or 25 μg/ml, grown in liquid culture for 1, 2, or 4-h, and plated on solid media for determination of colony forming units (CFUs). The fold change refers to CFUs at the 1, 2, and 4-h time points relative to Time = 0-h, which did not contain norfloxacin. Data are presented as mean values ± s.d. among three replicates. b. Immunoblot analysis of the membrane fraction after induction of MRSAΔnorA or MRSAΔnorA transformed with a hemin-inducible plasmid encoding wild-type NorA and NorA mutants. The samples were immunoblotted for NorA and SrtA (control membrane protein). Similar results were obtained in two immunoblots. c. Growth inhibition experiments using the MRSAΔnorA strain transformed with a hemin-inducible plasmid encoding NorA or NorA mutants. NorA expression was induced with 1 μM hemin and carried out in the presence or absence of 12.5 μg/mL norfloxacin. Solid lines and shading in the same color correspond to the average and standard deviation of four technical replicates from one representative experiment, respectively. Similar results were verified in at least two independent experiments. P-values were calculated using an unpaired, two-tailed t-test for each single-site mutant relative to MRSAΔnorA + NorA at the 10-h timepoint: ****P < 0.0001, ** P = 0.0045. d. Top: Representative serial dilution experiment on LB agar using E. coli transformed with a plasmid encoding NorA, NorA mutants, or no construct (vector) in the presence of norfloxacin (15 nM). Bottom: Results of serial dilution experiments for 20 single-site NorA mutants at aspartate and glutamate positions. Three mutants showed ablated resistance phenotypes toward norfloxacin (D63A, E222A, and D307A). The serial dilution screen was performed one time; loss-of-function mutants identified from the screen were confirmed in independent E. coli serial dilution experiments and in MRSA∆norA using MIC and growth inhibition experiments. e. Top: Representative images from norfloxacin MIC experiments using MRSAΔnorA strains transformed with a hemin-inducible plasmid encoding NorA (left) or D63A (right). NorA expression was induced with 1 μM hemin. The MIC values were read at the intersection of the inhibition eclipse and the strip (units of μg/ml). Bottom: Plot of norfloxacin MIC values for MRSA or MRSAΔnorA transformed with plasmids encoding wild-type NorA or NorA mutants. The MIC upper limit of detection was 256 μg/ml. P-values were calculated using an unpaired, two-tailed t-test for each single-site mutant relative to MRSAΔnorA + NorA: **** P < 0.0001. The number of independent MIC experiments (n = 2-5) correspond to the number of black circles superimposed on the bar graph for each sample; data are presented as mean values ± s.d. among the independent experiments.

Source data

Extended Data Fig. 8 Fab inhibition of NorA assessed through an E. coli co-expression assay.

a. Schematic illustrating Fab inhibition of NorA-mediated norfloxacin efflux. The AlphaFold model of inward-open NorA (https://alphafold.ebi.ac.uk/entry/Q2G0A2) is shown on the left and the cryo-EM structure of outward-open NorA from the NorA-Fab36 complex is displayed on the right. Efflux of norfloxacin is mediated by NorA in the absence of Fab (black arrow), whereas efflux is inhibited when NorA is bound to Fab (red ‘X’). b. Growth inhibition experiment of E. coli co-expressing NorA and Fabcontrol, Fab25, or Fab36. Experiments were performed in the presence (left) or absence (right) of 1.2 μg/mL norfloxacin and arabinose (inducer of Fabs; abbreviated ‘ara’). For clarity, only a few datasets at select arabinose concentrations are displayed. Solid lines and shading in the same color correspond to the average and standard deviation of four independent experiments, respectively. P-values were calculated using an unpaired, two-tailed t-test with Welch’s correction for the 24-h timepoint and indicate significance relative to the 0% arabinose concentration: **** P < 0.0001, ** P = 0.0013, * P = 0.0305. c. Analysis of the growth inhibition data at the 24-h timepoint for E. coli co-expressing NorA and Fabcontrol, Fab25, or Fab36 as a function of arabinose concentration. Bar graphs indicate mean values ± s.d. among four independent experiments, each with two replicates per experiment. Black circles correspond to all replicate datapoints from the independent experiments. d. Immunoblot analyses of E. coli co-expressing NorA and Fabcontrol, Fab25, or Fab36 after 24-h of growth and in the presence of varying arabinose concentration. Analyses were performed from growths in the absence of norfloxacin to normalize against growth inhibition variation when norfloxacin was present. Similar results were obtained in two immunoblots. e. Normalized E. coli growth curves calculated by dividing the OD600nm, 24-h value in the presence of norfloxacin by the corresponding value in the absence of norfloxacin for each arabinose concentration. Data are presented as mean values ± s.d. among four independent experiments. P-values were calculated using an unpaired, two-tailed t-test with Welch’s correction for the highest arabinose concentration for the NorA + Fab25 and NorA + Fab36 samples relative to the NorA + Fabcontrol sample: **** P < 0.0001, ** P = 0.0084. f. Immunoblot analysis of a NorA pull-down experiment for E. coli co-expressing NorA and Fabcontrol, Fab25, or Fab36 with 0.2% arabinose. Following co-expression, cells were lysed and NorA was purified from the membrane fraction using affinity chromatography. Elution fractions were immunoblotted for NorA and Fab. Similar results were obtained in two immunoblots.

Source data

Extended Data Fig. 9 Inhibition of MRSA growth by combination treatment of norfloxacin and a CDRH3 mimicking peptide targeting NorA.

a. Analytical HPLC traces (top row) and MALDI-TOF (bottom row) of purified NPI-1, NPI-2, and FITC-NPI-1 peptides. NPI-1: observed mass of 1802.05 [M + H]+; calculated mass of 1801.85 [M + H]+. NPI-2: observed mass of 1775.07 [M + H]+; calculated mass of 1774.79 [M + H]+. FITC-NPI-1: observed mass of 2263.76 [M + H]+; calculated mass of 2263.95 [M + H]+. b, c. Growth inhibition of MRSA or MRSAΔnorA treated with varying concentrations of (b) NPI-1 or (c) NPI-2 in the presence or absence of norfloxacin (12.5 μg/ml). The peptide was added at the start of the time course (Time = 0-h). Solid lines and shading in the same color correspond to the average and standard deviation of three independent experiments, respectively. d. Growth inhibition of MRSA or MRSAΔnorA at the 10-h timepoint from (b) and (c) plotted against the NPI-1 (left) or NPI-2 (right) concentration, respectively. Data are presented as mean values ± s.d. among three independent experiments. The fitted curve was used to determine the IC50 for NPI-1 (0.72 ± 0.08 μM).

Source data

Supplementary information

Supplementary Table 1

Cryo-EM data collection and structure determination statistics.

Reporting Summary

Supplementary Video 1

Three-dimensional variability analysis of the NorA–Fab25 complex map. NorA–Fab25 is initially oriented in the standard view shown in Fig. 1, with the N- and C-terminal domains of NorA on the left and right, respectively. The variable and constant domains of the Fab are denoted. The complex is rotated clockwise 360°, and an identical set of oscillations are displayed at each 90° interval.

Supplementary Video 2

Three-dimensional variability analysis of the NorA–Fab36 complex map. NorA–Fab36 is initially oriented in the standard view shown in Fig. 1, with the N- and C-terminal domains of NorA on the left and right, respectively. The variable and constant domains of the Fab are denoted. The complex is rotated clockwise 360°, and an identical set of oscillations are displayed at each 90° interval.

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Unprocessed western blots.

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Unprocessed western blots.

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Brawley, D.N., Sauer, D.B., Li, J. et al. Structural basis for inhibition of the drug efflux pump NorA from Staphylococcus aureus. Nat Chem Biol 18, 706–712 (2022). https://doi.org/10.1038/s41589-022-00994-9

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