Research ArticleBIOCHEMISTRY

Na+-induced structural transition of MotPS for stator assembly of the Bacillus flagellar motor

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Science Advances  01 Nov 2017:
Vol. 3, no. 11, eaao4119
DOI: 10.1126/sciadv.aao4119
  • Fig. 1 Schematic drawing of the bacterial flagellar motor.

    (A) Schematic diagram of the flagellar motor. The flagellar motor consists of a rotor ring complex and a dozen stator units made of two TM proteins, MotA and MotB. FliG, FliM, and FliN assemble into the C ring on the cytoplasmic face of the MS ring in this order and act as a rotor. (B) Schematic diagram of the MotAB complex. MotA and MotB together form a proton channel with four copies of MotA and two copies of MotB. Cα ribbon representation of the Salmonella MotBC structure (PDB code: 2ZVY) with the TM of MotAB and a flexible stalk of MotB (residues 51 to 100) connecting these two domains are shown. A highly conserved Asp33 residue (indicated as D) is directly involved in proton translocation through the MotAB proton channel complex. The stalk contains the plug segment (residues 53 to 66), which regulates the proton channel activity before stator assembly around the rotor.

  • Fig. 2 HS-AFM imaging of wild-type MotPS and its mutant derivatives.

    (A) Schematic diagram of the Na+-type MotPS complex. The MotPS complex is composed of four copies of MotP and two copies of MotS. The distance between the periplasmic surface of the cytoplasmic membrane and the inner surface of the peptidoglycan layer is about 10 nm. (B) Primary structures of Bacillus MotB, MotS, and various mutant variants of MotS. B. subtilis MotB and MotS have a single TM helix and a C-terminal large periplasmic domain containing a PGB motif termed an OmpA-like domain. MotPSΔperi and MotPSΔplug lack the indicated region of MotS. MotPSD30A has the D30A substitution at a putative Na+-binding site of MotS-TM. MotPSB-PGB has an OmpA-like domain of MotB instead of its original OmpA-like domain. aa, amino acid. (C) Typical HS-AFM images of wild-type MotPS, MotPSΔperi, MotPSB-PGB, MotPSΔplug, and MotPSD30A, placed on mica in a buffer containing 150 mM NaCl. All images were recorded at 200 ms per frame in a scanning area of 50 nm × 50 nm with 150 pixels × 150 pixels. Color bar shows a range of particle height (nanometers). Scale bars, 10 nm.

  • Fig. 3 Na+-induced structural transitions of the MotSC dimer.

    (A) Real-time imaging of MotPS with exchanging the salt in the buffer from 150 mM KCl to 150 mM NaCl. MotSC became suddenly folded after approximately 60 s (second and third panels) from the solvent exchange indicated as 0.00 s (0 mM NaCl in fig. S5C). (B) Real-time imaging of MotPS with exchanging the salt from 150 mM NaCl to 150 mM KCl. MotSC became suddenly unfolded after approximately 5 s (second and third panels) from the solvent exchange indicated as 0.00 s (150 mM NaCl in fig. S5D). All images in (A) and (B) were recorded at 250 ms per frame in a scanning area of 50 nm × 50 nm with 150 pixels × 150 pixels. Scale bars, 10 nm. (C) Effect of NaCl concentrations on the structural transitions of the PGB domain of MotSC. At least 100 individual particles were analyzed under each condition. The data points were fitted by the Hill’s equation.

  • Fig. 4 Correlation between the center-to-center distance between the small and large domains and the domain heights.

    Sequential HS-AFM images and their cross sections are shown for MotPS (A), MotPSΔplug (C), and MotPSD30A (E). All images were recorded at 200 ms per frame in a scanning area of 50 nm × 50 nm with 150 pixels × 150 pixels. Scale bars, 10 nm. Surface profiles along the line (from filled to open arrowheads) were fitted by two Gaussian functions, and the peak-to-peak distance and peak heights were measured. Distance versus height plots were generated for MotPS (B), MotPSΔplug (D), and MotPSD30A (F) from data points collected from more than 600 frames of 20 individual molecules. Blue and red dots are height data for the large and small domains, respectively. Histograms were fitted by multiple Gaussian functions.

  • Fig. 5 Model for the Na+-induced assembly and disassembly of the MotPS stator.

    Structural transitions of MotSC between the unfolded and folded states occur in a Na+-dependent manner. The plug region of MotSC is detached from the TM Na+ channel formed by the TM helices of MotP and MotS, followed by partial unfolding of the N-terminal portion of the PGB domain of MotSC, causing a 5-nm extension of the PGB domain from the TM domain of MotPS to reach the peptidoglycan layer, allowing MotPS to become an active Na+-type stator unit.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/11/eaao4119/DC1

    fig. S1. Primary structures of MotB and its homologs, MotS and PomB.

    fig. S2. Effect of Na+ concentrations on motor rotation of the flagellar motor in wild-type Bacillus cells expressing both MotAB and MotPS.

    fig. S3. Purification of His6-tagged MotPS by size exclusion chromatography.

    fig. S4. Comparison of simulated AFM images of the MotBC and the MotA tetramer with experimental image of the MotPS complex.

    fig. S5. Two distinct conformations of the PGB domain of MotS.

    fig. S6. Motility of motS mutants.

    table S1. Rotational speed and torque of the wild-type motor.

    table S2. Speed fluctuations of the wild-type, MotAB, and MotPS motor.

    movie S1. Real-time imaging of wild-type MotPS by HS-AFM.

    movie S2. Typical HS-AFM imaging of wild-type MotPS in buffer containing 150 mM NaCl.

    movie S3. Typical HS-AFM imaging of MotPSΔperi in buffer containing 150 mM NaCl.

    movie S4. Typical HS-AFM imaging of MotPSB-PGB in buffer containing 150 mM NaCl.

    movie S5. Real-time imaging of a disorder-to-order transition of MotPS with an increase in the concentration of NaCl.

    movie S6. Real-time imaging of a order-to-disorder transition of MotPS with a decrease in the concentration of NaCl.

    movie S7. Typical HS-AFM imaging of MotPSΔplug in buffer containing 150 mM NaCl.

    movie S8. Typical HS-AFM imaging of MotPSD30A in buffer containing 150 mM NaCl.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Primary structures of MotB and its homologs, MotS and PomB.
    • fig. S2. Effect of Na+ concentrations on motor rotation of the flagellar motor in wild-type Bacillus cells expressing both MotAB and MotPS.
    • fig. S3. Purification of His6-tagged MotPS by size exclusion chromatography.
    • fig. S4. Comparison of simulated AFM images of the MotBC and the MotA tetramer with experimental image of the MotPS complex.
    • fig. S5. Two distinct conformations of the PGB domain of MotS.
    • fig. S6. Motility of motS mutants.
    • table S1. Rotational speed and torque of the wild-type motor.
    • table S2. Speed fluctuations of the wild-type, MotAB, and MotPS motor.
    • Legends for movies S1 to S8

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mov format). Real-time imaging of wild-type MotPS by HS-AFM.
    • movie S2 (.mov format). Typical HS-AFM imaging of wild-type MotPS in buffer containing 150 mM NaCl.
    • movie S3 (.mov format). Typical HS-AFM imaging of MotPSΔperi in buffer containing 150 mM NaCl.
    • movie S4 (.mov format). Typical HS-AFM imaging of MotPSB-PGB in buffer containing 150 mM NaCl.
    • movie S5 (.mov format). Real-time imaging of a disorder-to-order transition of MotPS with an increase in the concentration of NaCl.
    • movie S6 (.mov format). Real-time imaging of a order-to-disorder transition of MotPS with a decrease in the concentration of NaCl.
    • movie S7 (.mov format). Typical HS-AFM imaging of MotPSΔplug in buffer containing 150 mM NaCl.
    • movie S8 (.mov format). Typical HS-AFM imaging of MotPSD30A in buffer containing 150 mM NaCl.

    Files in this Data Supplement:

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