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Metastable asymmetrical structure of a shaftless V1 motor

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Science Advances  30 Jan 2019:
Vol. 5, no. 1, eaau8149
DOI: 10.1126/sciadv.aau8149
  • Fig. 1 Crystal structures of the A3B3 and A3B3DF (V1) complexes.

    (A) Side view of the nucleotide-free A3B3 (eA3B3) structure divided into three domains as shown with two lines: N-terminal [residues A (1–71) and B (1–77)], central [residues A (72–449) and B (78–362)], and C-terminal [residues A (450–593) and B (363–458)] domains. A and B subunits are depicted in blue and purple, respectively. (B to F) Top views of N-terminal β-barrel domain (crown) (B), central domain (C), and C-terminal domains of eA3B3 (D), AMP-PNP bound A3B3 (bA3B3) (E), and AMP-PNP bound V1 (bV1) (F) as surface representations viewed from the N-terminal β-barrel side. Red arrows indicate the nucleotide binding sites. The bound AMP-PNP molecules are shown as red spheres. The DF shaft is shown in green.

  • Fig. 2 HS-AFM images of the wild-type and mutant A3B3 complexes.

    (A) Schematic image of HS-AFM imaging observation of A3B3 from the side with the N-terminal domain at the bottom. A and B subunits are shown in blue and purple, respectively, as a surface representation. The tip of the short cantilever is also drawn. (B and C) HS-AFM images of wild-type A3B3 (B) and mutant A3B(L65Y)3 (C) showing 50 nm × 50 nm area. The insets (30 nm × 8 nm) are the height line profiles from the path indicated by a white line in the images with the height of the highest peak marked. (D and E) Five representative HS-AFM images of wild-type A3B3 as averaged images, with the contrast of the height color table adjusted (see Materials and Methods). An A subunit (higher subunit) and a B subunit (lower subunit) are indicated at #1 in (D). The orientations of averaged images were adjusted to be the same for all molecules. Scale bar is indicated by a black bar and denotes 5 nm. (F and G) Five representative averaged images of the mutant A3B(L65Y)3. Zero nanometer of the color maps in (B) and (C) is the lowest point of the images, whereas in (D) to (G) it is the highest.

  • Fig. 3 Crystal structure of nucleotide-free A3B(L65Y)3.

    (A) Top view of N-terminal β-barrel (or crown) structure shown as in Fig. 1B. The mutated residues are shown in yellow. (B) Top view of the central domain of eA3B(L65Y)3 shown as in Fig. 1C. (C) Top view of the C-terminal domain of eA3B(L65Y)3 as in Fig. 1D, which is superimposed at the N-terminal β-barrel domain of the bindable form (chains B and E) of A1B(L65Y)1 units onto that of eA3B3 (gray). (D and E) Superimposed structures using Cα atoms of three A (blue) and B (purple) subunits in eA3B(L65Y)3. The P-loop and arm are shown in yellow and white, respectively. The green box indicates the nucleotide binding site. (F) Magnified views of the nucleotide binding sites with conserved residues, corresponding to the green box in (E).

  • Fig. 4 Crystal structure of AMP-PNP bound A3B(L65Y)3.

    (A) Side-top view of AMP-PNP bound mutant A3B(L65Y)3 [bA3B(L65Y)3]. N-terminal domain is shown in surface representation, and the central and C-terminal domains are in cartoon representations. Colors are the same as in Fig. 1E. (B and C) Top views of the N-terminal (B) and central (C) domains as in Fig. 3, A and B, respectively. (D) Top view of the C-terminal domain of bA3B(L65Y)3 as in Fig. 1E. (E to G) Magnified views of the nucleotide binding sites. The sites corresponding to the green box in Fig. 3E are shown for bA3B(L65Y)3. The P-loop and arm are shown in yellow and white, respectively. The right panels show the A-B interface rotated 90° around a vertical axis from the left panels, with conserved residues of the “bound” (E), “tight(1)” (F), and “tight(2)” (G) forms in bA3B(L65Y)3. To compare structural differences, two dotted vertical lines were drawn from the Cα position of R350 in bound form (E) and tight forms (F and G) to show the closer location (approximately 1.2 Å) of the tight form to the bound nucleotides. The |Fo| − |Fc| map calculated without AMP-PNP: Mg2+ at the binding pockets at 4.0σ are shown in red (negative) and green (positive) meshes.

  • Fig. 5 Schematic structure models of the wild-type and mutant A3B3.

    The A3B3 complexes are pictured as a side view from the inside, with the ring cut and laid flat so that all the subunits can be viewed simultaneously. A and B subunits are colored in blue and purple, respectively. The crown is highlighted by a light yellow box. The dotted lines signify where the ends would connect to form the ring. Mutant (A and C) and wild-type A3B3 (B and D) models are drawn showing the conformational changes induced by the nucleotide binding [from (A) to (C) and (B) to (D), respectively]. Yellow half-circles show the mutated amino acid, L65Y, in the crown. Corresponding to the weak crown, mutant models show a convex crown (A) and a concave broken crown (C). AMP-PNP molecules are shown as black squares. The nucleotide binding sites are depicted as white square boxes that are open, half-filled, or filled. The empty form has a specifically low affinity for nucleotides. Therefore, the square boxes indicating the binding sites are distorted to look like a trapezoid. The form name is presented under each A1B1 unit with a description of the preferable (or stable) form or the less stable form.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/1/eaau8149/DC1

    Fig. S1. Purification and ATPase activity of wild-type A3B3, mutant A3B(L65Y)3, and DF complexes.

    Fig. S2. Electron micrographs of negatively stained wild-type and mutant A3B3 complexes.

    Fig. S3. Comparison of the crown structures of wild-type and mutant A3B3 complexes.

    Fig. S4. Magnified views of the nucleotide binding sites of the bindable forms in eA3B(L65Y)3.

    Fig. S5. Comparison of the nucleotide binding sites of wild-type and mutant complexes.

    Table S1. Data collection and refinement statistics.

    Table S2. RMSD values in superimpositions for each A1B1 unit of eA3B(L65Y)3 in the crystal structures of A3B3 and V1 complexes.

    Table S3. Contact surface areas between A and B subunits in various A1B1 forms (Å2).

    Table S4. Sum of contact surface areas at the central domain in A3B32).

    Table S5. RMSD values in superimpositions for each A1B1 unit of bA3B(L65Y)3 in the crystal structures of A3B3 and V1 complexes.

    Movie S1. AFM scan movie of the C-terminal side of the wild-type and mutant A3B3.

    Movie S2. Conformational changes at the β-barrel domain induced by the mutation.

    Movie S3. Conformational changes at the C-terminal domain of eA3B3 induced by the mutation.

    Movie S4. Conformational changes of eA3B(L65Y)3 induced by binding AMP-PNP molecules.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Purification and ATPase activity of wild-type A3B3, mutant A3B(L65Y)3, and DF complexes.
    • Fig. S2. Electron micrographs of negatively stained wild-type and mutant A3B3 complexes.
    • Fig. S3. Comparison of the crown structures of wild-type and mutant A3B3 complexes.
    • Fig. S4. Magnified views of the nucleotide binding sites of the bindable forms in eA3B(L65Y)3.
    • Fig. S5. Comparison of the nucleotide binding sites of wild-type and mutant complexes.
    • Table S1. Data collection and refinement statistics.
    • Table S2. RMSD values in superimpositions for each A1B1 unit of eA3B(L65Y)3 in the crystal structures of A3B3 and V1 complexes.
    • Table S3. Contact surface areas between A and B subunits in various A1B1 forms (Å2).
    • Table S4. Sum of contact surface areas at the central domain in A3B32).
    • Table S5. RMSD values in superimpositions for each A1B1 unit of bA3B(L65Y)3 in the crystal structures of A3B3 and V1 complexes.
    • Legends for movies S1 to S4

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). AFM scan movie of the C-terminal side of the wild-type and mutant A3B3.
    • Movie S2 (.mov format). Conformational changes at the β-barrel domain induced by the mutation.
    • Movie S3 (.mov format). Conformational changes at the C-terminal domain of eA3B3 induced by the mutation.
    • Movie S4 (.mov format). Conformational changes of eA3B(L65Y)3 induced by binding AMP-PNP molecules.

    Files in this Data Supplement:

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