Research ArticleSTRUCTURAL BIOLOGY

Pathogenic siderophore ABC importer YbtPQ adopts a surprising fold of exporter

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Science Advances  05 Feb 2020:
Vol. 6, no. 6, eaay7997
DOI: 10.1126/sciadv.aay7997
  • Fig. 1 Functional characterization of YbtPQ.

    (A) Ybt-Fe uptake experiments. The relative amount of Ybt-Fe remaining in the solution after uptake by E. coli cells with either empty vector (dotted line, negative control) or overexpressed YbtPQ (solid line) is plotted against time. (B) ATP hydrolysis activity of YbtPQ-LMNG (top) and YbtPQ-nanodisc (bottom) plotted against ATP concentration. For both samples, Vmax and Km are not distinguishable at ~10 nmol mg−1 min−1 and ~0.5 mM, respectively. The error bars and SD are from the means of three independent experiments. (C) MST analysis of Ybt-Fe binding to the YbtPQ samples. Both YbtPQ-LMNG and YbtPQ-nanodisc have similar Kd values when binding the substrate.

  • Fig. 2 Cryo-EM analysis of YbtPQ and its overall architecture.

    (A) The atomic model of YbtPQ-LMNG fits in its cryo-EM map. YbtP is in blue, YbtQ is in pink, while the cryo-EM density is in gray. The dotted black circle indicates a protruded density, probably consisting of copurified lipids. (B) A tunnel open to the cytoplasm with a minimum diameter of 1.5 Å is shown in green mesh, with a substrate molecule, Ybt-Fe, in dark gray sitting in the substrate-binding pocket. Zoom-in of this same view of the substrate-binding pocket is shown in Fig. 3. (C) Superposition of YbtP and YbtQ. Six TMs and two ICLs are labeled. When the two proteins are aligned with TMDs, their NBDs appear to be 10° rotated away.

  • Fig. 3 Network of interactions in the substrate-binding pocket.

    (A) Important hydrophobic interactions between Ybt-Fe and YbtPQ in front (left panel) and side (right panel) views. Four helices forming the pocket are shown as ribbon, and five aromatic residues are shown as stick. (B) Important hydrogen bonds in the substrate-binding pocket in front (left panel) and top (right panel) views. Hydrogen bonds formed between residues and Ybt-Fe are shown as dotted lines. YbtP residues are in blue, and YbtQ residues are in pink. Ybt-Fe is colored by heteroatom, with the Fe molecule in orange.

  • Fig. 4 YbtP-TM4 unwinding upon substrate release.

    (A) Superposition of YbtP from YbtPQ structures with and without Ybt-Fe. YbtP with Ybt-Fe is in blue, while YbtP without Ybt-Fe is in gray. (B) Model (ribbon) and map (green mesh) of the isolated YbtP-TM4 region from both YbtPQ structures.

  • Fig. 5 NBDs in YbtPQ.

    (A) Interface between TMDs and NBDs in front and top views. NBDs are in gray, with YbtP shown as ribbon and YbtQ shown as surface. The interacting loops in NBDs are in orange (A-loop) and black (Q-loop). ICLs are shown as ribbon. (B) ATP binding sites and conserved motifs in top view. Walker A motif is in red, ABC signature motif is in green, and D-loop is in purple. The distance between Cα atoms of conserved residues from Walker A, ABC, and D-loop is indicated by dotted lines. (C) Association between NBDs in bottom view. Important residues for hydrophobic interactions between helices are highlighted in blue.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/6/eaay7997/DC1

    Fig. S1. Purification of YbtPQ in LMNG.

    Fig. S2. Negative-stain EM of YbtPQ.

    Fig. S3. Cryo-EM data quality of YbtPQ.

    Fig. S4. Cryo-EM reconstruction of YbtPQ.

    Fig. S5. Cryo-EM reconstruction of YbtPQ-nanodisc.

    Fig. S6. Cryo-EM reconstruction of YbtPQ-LMNG-Ybt-Fe.

    Fig. S7. MALDI-MS analysis of YbtPQ-LMNG.

    Fig. S8. Ybt-Fe substrate density in cryo-EM map.

    Fig. S9. Sequence alignment of YbtPQ with homologs.

    Table S1. Cryo-EM data collection, refinement, and validation statistics.

    Table S2. Substrate-binding affinity of YbtPQ mutagenesis measured by MST.

    Table S3. Distances between key conserved motifs in various ABC transporters.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Purification of YbtPQ in LMNG.
    • Fig. S2. Negative-stain EM of YbtPQ.
    • Fig. S3. Cryo-EM data quality of YbtPQ.
    • Fig. S4. Cryo-EM reconstruction of YbtPQ.
    • Fig. S5. Cryo-EM reconstruction of YbtPQ-nanodisc.
    • Fig. S6. Cryo-EM reconstruction of YbtPQ-LMNG-Ybt-Fe.
    • Fig. S7. MALDI-MS analysis of YbtPQ-LMNG.
    • Fig. S8. Ybt-Fe substrate density in cryo-EM map.
    • Fig. S9. Sequence alignment of YbtPQ with homologs.
    • Table S1. Cryo-EM data collection, refinement, and validation statistics.
    • Table S2. Substrate-binding affinity of YbtPQ mutagenesis measured by MST.
    • Table S3. Distances between key conserved motifs in various ABC transporters.

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