Research ArticleBIOCHEMISTRY

Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline

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Science Advances  08 May 2015:
Vol. 1, no. 4, e1500106
DOI: 10.1126/sciadv.1500106
  • Fig. 1 Amino acid alignment of selected ATP synthase c-subunits.

    The location of the N- and C-terminal helices and the loop region (bold letters) are indicated (top). Amino acid numbering (top) is according to M. phlei. Amino acids previously shown to cause a BDQ resistance upon mutation (see Results and Discussion section for details) are indicated in red; amino acids at the same position but from other species are shaded in gray. Residues found to be involved in drug coordination, based on the x-ray structure, are highlighted in blue. The ion-binding glutamate is indicated by an arrow. M. tb. H37Rv, Mycobacterium tuberculosis H37Rv; M. smeg., Mycobacterium smegmatis; M. fort., Mycobacterium fortuitum; M. absc., Mycobacterium abscessus; Sp. chl., spinach chloroplast; S. plat., Spirulina platensis; I. tart., Ilyobacter tartaricus; F. nuc., Fusobacterium nucleatum.

  • Fig. 2 Inhibition of ATP synthesis of M. phlei F1Fo-ATP synthase by BDQ.

    (A) Continuous ATP synthesis of M. phlei IMVs (50 μg) monitored by increase in luminescence (blue). The presence of 0.1 μM BDQ (red) immediately and completely abolishes the synthesis of ATP. Negative controls: uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCCP), ATP synthase inhibitor dicyclohexylcarbodiimide (DCCD), and no ADP. (B) Inhibition of ATP synthesis versus BDQ concentrations of 0 to 1 μM. (C) Zoom of the marked area (0 to 0.1 μM) in (B). The data were used to calculate an IC50 value of 20 to 25 nM. For details, see Materials and Methods.

  • Fig. 3 BDQ binding to the isolated M. phlei c-ring using an inhibitor competition assay.

    Purified samples of M. phlei c-ring (0.1 mg/ml) were preincubated with 0 to 30 μM BDQ, and the time-dependent formation of DCCD-modified c-subunits was determined. (A) MALDI mass spectra of c-subunits after incubation with DCCD in the absence (left panel) or presence of 10 μM BDQ (right panel) after 5 min (top) or 30 min (bottom). Unmodified c-subunits are indicated by black diamonds (♦), and dicyclohexyl-N-acylisourea (DCU)–modified c-monomers are indicated by asterisks (*). BDQ (10 μM) efficiently blocks binding of DCCD to c-monomers as indicated by the yellow arrows in the right panel [no signal in corresponding mass/charge ratio (m/z) range]. (B) Statistics of % DCCD labeling after preincubation of the c-ring with BDQ concentrations 0 to 30 μM. A markedly reduced DCCD labeling efficacy was observed upon preincubation with BDQ, indicating that both inhibitors compete for the same binding site. The SD was calculated from at least three individual experiments. For details, see Materials and Methods.

  • Fig. 4 Structure of the M. phlei c9 ring without and in complex with BDQ.

    (A) The c9 ring with BDQ bound; Side view. (B) Top view of the c-ring (cartoon representation) with bound BDQ molecules (black). Membrane borders (gray bars) and water molecules (red spheres) are indicated. (C) Slanted view of the ion-binding side showing the interaction of BDQ (2Fobs-Fcalc maps in black, at 1.1σ) with the c-ring. The anomalous difference map of the BDQ bromine is shown in red mesh at 4σ. Selected residues and bonding distances in angstrom (dashed lines) are indicated. BDQ invades into the ion-binding site with its dimethylamino (DMA) moiety and forms a specific ionic intermolecular H-bond with Glu65 (see the text). (D) Structure of the c9 ring without BDQ bound; side view of the ion-binding site. 2Fobs-Fcalc maps (gray mesh) are shown at 1.3σ. (E) Two-dimensional (2D) plot of the BDQ/c-ring interactions. Interaction distances are color-coded. Numerous van der Waals (VdW) interactions and two hydrogen bonds contribute directly to the highly specific binding of BDQ to the c-ring (see also table S2).

  • Fig. 5 Surface of the M. phlei c9 ring and electrostatic potential distribution.

    (A) Surface and electrostatic potential distribution of the M. phlei c9 ring. Membrane borders are indicated by gray bars. BDQ molecules are shown in black. (B) Surface comparison of the drug-binding region of the M. phlei c-ring with a M. tuberculosis c-ring homology model (generated using WHAT IF) (51), the I. tartaricus c11 ring (9), and the S. cerevisiae c10 ring (19). In the M. phlei and M. tuberculosis c-rings, the BDQ fits the ion-binding region, with the quinoline moiety sitting on the Phe platform (arrow) facilitating numerous interactions (see the text). In contrast, in the eukaryotic S. cerevisiae and the bacterial I. tartaricus c-rings, the Phe platform is missing (black circle) and the surface-determining side chains (dotted blue line) cause steric clashes.

  • Fig. 6 Structural alignment of BDQ-free and BDQ-bound M. phlei c-ring structures and start and end states of the drug binding process.

    (A and B) Side (A) and top (B) views (from cytoplasmic side) of the BDQ-free (green) and BDQ-bound (blue) M. phlei c-ring structures in cartoon representation, illustrating the conformational changes and water (green and blue spheres) rearrangements occurring upon BDQ (stick model, dark gray) binding. Red arrows indicate a movement of Phe69. Red circles indicate sites of overlapping regions between the BDQ-bound and BDQ-free structures that undergo conformational changes.

  • Fig. 7 Inhibition mechanism of BDQ.

    The mycobacterial ATP synthase Fo motor unit is shown from the cytoplasmic side, cut open at the level of the c-ring ion-binding sites. One or more (four examples are shown in black) BDQ molecules approach the c-ring surface from the hydrophobic zone of the lipid bilayer (gray area) [1]. Each BDQ molecule binds to the region of the ion-binding site and interacts with one of the conserved glutamate residues (Glu65 in M. phlei). The DMA group contacts the carboxyl group of the ion-binding glutamate. The bulky drug molecule bound to the c-ring is sterically and energetically disfavored to pass the a/c-ring interface: the rotor motion stalls [2], ion exchange at the ATP synthase [3], and finally ATP synthesis activity stops.

  • Table 1 Table of crystallography.
    c9 (4v1g)BDQ-bound c9 (4v1f)
    Data processing
    Wavelength (Å)0.99990.91747
    Space groupR 3 :HR 3 :H
    Cell dimensions
      a, b, c (Å)73.7, 73.7, 166.275.0, 75.0, 166.6
      α, β, γ (°)90, 90, 12090, 90, 120
    Resolution (Å)36.8–1.55 (1.6–1.55)37.5–1.7 (1.76–1.7)
    Number of observed reflections162,379 (11,775)392,443 (38,342)
    Number of unique reflections48,446 (4,618)38,464 (3,832)
    Redundancy3.4 (2.5)10.2 (10.0)
    Completeness (%)99.2 (94.6)99.46 (99.38)
    Rmrgd-F (%)4.5 (66.4)8.13 (58.33)
    II13.83 (1.44)20.38 (4.82)
    Refinement statistics
    Resolution (Å)36.8–1.5537.5–1.7
    Rwork/Rfree (%)15.66/17.8715.63/16.19
    Number of atoms1,9632,091
      Protein1,8231,828
      Ligands60163
      Water8098
      B-factors32.924.90
      Protein30.5021.5
      Ligands93.556.9
      Solvent42.135.1
    Root mean square deviations
      Bond length (Å)0.0200.007
      Bond angles (°)1.7651.09

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/4/e1500106/DC1

    Fig. S1. Comparison of the BDQ-free M. phlei c-ring ion-binding site with the c-ring ion-binding site from F. nucleatum.

    Fig. S2. Comparison of c-ring surface and electrostatic potential distribution.

    Fig. S3. Structural alignment and comparison of BDQ binding on the c-rings of a non-mycobacterial bacterium (I. tartaricus) and a eukaryotic, human homolog model (S. cerevisiae).

    Fig. S4. Comparison of the protein-bound and soluble, energy-minimized BDQ conformation.

    Table S1. BDQ minimum inhibitory concentrations.

    Table S2. Interactions of BDQ with surrounding amino acid residues and water molecules.

    Movie S1. Morph between the BDQ-free and BDQ-bound M. phlei c-ring structures viewed from the membrane.

    Movie S2. Morph between the BDQ-free and BDQ-bound M. phlei c-ring structures viewed from the cytoplasm.

    References (52, 53)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Comparison of the BDQ-free M. phlei c-ring ion-binding site with the c-ring ion-binding site from F. nucleatum.
    • Fig. S2. Comparison of c-ring surface and electrostatic potential distribution.
    • Fig. S3. Structural alignment and comparison of BDQ binding on the c-rings of a non-mycobacterial bacterium (I. tartaricus) and a eukaryotic, human homolog model (S. cerevisiae).
    • Fig. S4. Comparison of the protein-bound and soluble, energy-minimized BDQ conformation.
    • Table S1. BDQ minimum inhibitory concentrations.
    • Table S2. Interactions of BDQ with surrounding amino acid residues and water molecules.
    • Legend for movies S1 and S2
    • References (52, 53)

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1. Morph between the BDQ-free and BDQ-bound M. phlei c-ring structures viewed from the membrane.
    • Movie S2. Morph between the BDQ-free and BDQ-bound M. phlei c-ring structures viewed from the cytoplasm.

    Files in this Data Supplement:

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