Research ArticleSTRUCTURAL BIOLOGY

Insight into structural remodeling of the FlhA ring responsible for bacterial flagellar type III protein export

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Science Advances  25 Apr 2018:
Vol. 4, no. 4, eaao7054
DOI: 10.1126/sciadv.aao7054
  • Fig. 1 FlhAC forms a ring-shaped structure.

    (A) Typical HS-AFM image of FlhAC placed on mica at a protein concentration of 2.4 μM. The image was recorded at 200 ms per frame in a scanning area of 100 × 100 nm2 with 100 × 100 pixels. (B) Comparison of a simulated HS-AFM image (middle panel) constructed from the atomic model of the FlhAC9 ring structure (left panel) with an experimental HS-AFM image of the FlhAC ring (right panel) obtained at a concentration of 0.71 μM. The HS-AFM image was recorded at 200 ms per frame in a scanning area of 50 × 50 nm2 with 100 × 100 pixels. Color bar shows a range of particle height (nanometers). (C to E) Histograms of the stoichiometry (C), diameter (peak-to-peak distance), (D) and height (E) of the FlhAC ring. (F) Effect of protein concentrations on FlhAC ring formation. Ring particles per frame were counted under each condition. The number of the ring formed at the highest protein concentration is set to 1. Relative ring particle numbers versus protein concentrations (micromolar) are plotted and fitted by Hill equation.

  • Fig. 2 Effect of deletion of residues 328 to 351 in FlhAL on FlhAC ring formation.

    (A) Crystal structure of the FlhAC dimer [PDB (Protein Data Base) ID: 3A5I]. FlhAC contains four domains, D1, D2, D3, and D4, and a flexible linker termed FlhAL. The asymmetric unit of the crystal contains two FlhAC molecules. Residues 347 to 361 of FlhAL bind to the D1 and D3 domains of its neighboring subunit. Residues involved in this interaction are shown. The Cα backbone is color-coded from blue to red, going through the rainbow colors from the N terminus to the C terminus. (B) Typical HS-AFM images of wild-type FlhAC (indicated as WT) and FlhAC38K (indicated as 38K) lacking residues 328 to 351 of FlhAL placed either on mica or mica-supported planar phospholipid bilayer (mica-SLB). The protein concentrations were ca. 2.4 and 11.8 μM when they were placed on the mica and phospholipid surfaces, respectively. The HS-AFM images on the mica surface were recorded at 200 ms per frame in a scanning area of 100 × 100 nm2 with 150 × 150 pixels. The images on phospholipids were recorded at 500 ms per frame in a scanning area of 100 × 100 nm2 with 200 × 200 pixels. Scale bars, 10 nm. (C) Effect of protein concentrations on FlhAC ring formation on phospholipid bilayer. Ring particles per frame were counted under each condition. Relative ring particle numbers versus protein concentrations (micromolar) are plotted and fitted by Hill equation. (D) Typical HS-AFM images of wild-type FlhAC and its mutant variants, W354A, E351A/D356A, E351A/W354A/D356A, R391A/K392A/K393A, and L401A placed on a mica surface at a concentration of 2.4 μM. All images were recorded at 200 ms per frame in a scanning area of 100 × 100 nm2 with 150 × 150 pixels. Scale bars, 10 nm.

  • Fig. 3 Effect of alanine substitutions of residues involved in interactions of FlhAL with its neighboring subunit on FlhA function.

    (A) Secretion assays of flagellar proteins. Immunoblotting using polyclonal anti-FlgD (first row), anti-FlgE (second row), anti-FlgM (third row), anti-FlgK (fourth row), anti-FlgL (fifth row), or anti-FliC (sixth row) antibody, of whole-cell proteins (Cell) and culture supernatants (Sup). Lanes 1 and 9, ΔflhA; lanes 2 and 10, wild-type (WT); lanes 3 and 11, flhA(W354A) (W354A); lanes 4 and 12, flhA(E351A/D356A) (E351A/D356A); lanes 5 and 13, flhA(E351A/W354A/D356A) (E351A/W354A/D356A); lanes 6 and 14, flhA(R391A/K392A/K393A) (R391A/K392A/K393A); lanes 7 and 15, flhA(L401A) (L401A); and lanes 8 and 16, flhA(Q511A) (Q511A). The positions of FlgD, FlgE, FlgM, FlgK, FlgL, and FliC are indicated by arrows. (B) Electron micrograms of HBB isolated from the above stains. The average hook length and SDs are shown. Scale bars, 100 nm. (C) Interaction between FlhAC and FlgN-FlgK complex. Purified His-FlhAC (WT) or His-FlhAC(W354A) (W354A) was mixed with GST-FlgN in complex with FlgK (first and second rows), or GST alone (third row) and dialyzed overnight against phosphate-buffered saline (PBS) and dialyzed overnight against PBS. The mixture (L) was subjected to GST affinity chromatography. Flow through fraction (F.T.), wash fractions (W), and elution fractions (E) were analyzed by CBB (Coomassie brilliant blue) staining.

  • Fig. 4 Effect of depletions of FliH and FliI on the export function of FlhA(L401A).

    (A) Interaction between FliJ and FlhAC. The mixture of the soluble fractions (Input) prepared from Salmonella ΔflhDC-cheW cells expressing GST-FliJ with those from a Salmonella ΔflhA mutant carrying pTrc99AFF4 (indicated as ΔflhA), pMM130 (WT), pYI001 (W354A), pYI002 (E351A/D356A), pYI003 (E351A/W354A/D356A), pYI004 (R391A/K392A/K393A), pYI005 (L401A), or pYI006 (Q511A) was loaded onto a GST column. After extensive washing, proteins were eluted with a buffer containing 10 mM reduced glutathione. The eluted fractions were analyzed by immunoblotting with polyclonal anti-FlhA antibody (first and second rows) and CBB staining for GST-FliJ (third row). (B) Motility of and flagellar protein export by NH004 (ΔfliH-fliI flhB* ΔflhA) (left panel) and NH002 (flhB* ΔflhA) (right panel) harboring pTrc99AFF4 (V), pMM130 (WT), pYI005 (L401A), or pYI006 (Q511A). Upper panels: Motility assays in soft agar at 30°C. Lower panels: Immunoblotting, using polyclonal anti-FlgD and anti-FlgK antibodies, of whole-cell proteins (Cell) and culture supernatant fractions (Sup).

  • Fig. 5 Model for cooperative remodeling of the FlhAC ring structure.

    Partial atomic models of the MxiAC (left; PDB ID: 4A5P) and FlhAC (right; PDB ID: 3A5I) ring models are shown. Both MxiAC and FlhAC consist of domains D1 (cyan), D2 (blue), D3 (light green), and D4 (red). FlhAC forms a nonameric ring structure mainly through interactions between D3 domains and between domains D1 and D3. Only an N-terminal short stretch forming part (magenta) of a flexible linker is visible, whereas most of residues forming the linker are invisible because of their conformational flexibility. In contrast, about a C-terminal half of FlhAL binds to its neighboring subunit in the FlhAC crystal. During hook assembly, FlhAL does not bind to its neighboring subunit, allowing the flagellar type III protein export apparatus to transport the hook protein. Upon completion of the hook structure, FlhAL binds to the D1 and D3 domains of its neighboring subunit, inducing conformational changes of the FlhAC ring in a highly cooperative manner. As a result, the protein export apparatus terminates the export of the hook protein and initiates the export of filament-type proteins responsible for filament formation at the hook tip.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/4/eaao7054/DC1

    fig. S1. Comparison of experimental HS-AFM image of an FlhAC monomer with a simulated AFM image of the crystal structure of FlhAC.

    fig. S2. Effect of phospholipids on FlhAC ring formation.

    fig. S3. Effect on motility and flagellar formation of alanine substitutions of residues involved in interactions of FlhAL with its neighboring subunit in the FlhAC crystal.

    fig. S4. Effect of phospholipids on ring formation of FlhAC mutant variants.

    fig. S5. Effect of FlhA mutations on interactions of FlhAC with FlgN and FliJ.

    fig. S6. Effect of depletion of FliH and FliI on the export function of FlhA(L401A).

    fig. S7. Effect of the FlhA(F459A) mutation on FlhAC ring formation.

    table S1. Salmonella strains and plasmids used in this study.

    movie S1. Real-time imaging of FlhAC ring assembly on mica.

    movie S2. Typical HS-AFM imaging of wild-type FlhAC placed on mica surface.

    movie S3. Typical HS-AFM imaging of FlhAC38K placed on mica surface.

    movie S4. Typical HS-AFM imaging of wild-type FlhAC placed on mica-supported planar phospholipid bilayers.

    movie S5. Typical HS-AFM imaging of FlhAC38K placed on mica-supported planar phospholipid bilayers.

    References (4045)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Comparison of experimental HS-AFM image of an FlhAC monomer with a simulated AFM image of the crystal structure of FlhAC.
    • fig. S2. Effect of phospholipids on FlhAC ring formation.
    • fig. S3. Effect on motility and flagellar formation of alanine substitutions of residues involved in interactions of FlhAL with its neighboring subunit in the FlhAC crystal.
    • fig. S4. Effect of phospholipids on ring formation of FlhAC mutant variants.
    • fig. S5. Effect of FlhA mutations on interactions of FlhAC with FlgN and FliJ.
    • fig. S6. Effect of depletion of FliH and FliI on the export function of FlhA(L401A).
    • fig. S7. Effect of the FlhA(F459A) mutation on FlhAC ring formation.
    • table S1. Salmonella strains and plasmids used in this study.
    • Legends for movies S1 to S5
    • References (40–45)

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mov format). Real-time imaging of FlhAC ring assembly on mica.
    • movie S2 (.mov format). Typical HS-AFM imaging of wild-type FlhAC placed on mica surface.
    • movie S3 (.mov format). Typical HS-AFM imaging of FlhAC38K placed on mica surface.
    • movie S4 (.mov format). Typical HS-AFM imaging of wild-type FlhAC placed on mica-supported planar phospholipid bilayers.
    • movie S5 (.mov format). Typical HS-AFM imaging of FlhAC38K placed on mica-supported planar phospholipid bilayers.

    Files in this Data Supplement:

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