Atomic structures of a bactericidal contractile nanotube in its pre- and postcontraction states

Abstract

R-type pyocins are representatives of contractile ejection systems, a class of biological nanomachines that includes, among others, the bacterial type VI secretion system (T6SS) and contractile bacteriophage tails. We report atomic models of the Pseudomonas aeruginosa precontraction pyocin sheath and tube, and the postcontraction sheath, obtained by cryo-EM at 3.5-Å and 3.9-Å resolutions, respectively. The central channel of the tube is negatively charged, in contrast to the neutral and positive counterparts in T6SSs and phage tails. The sheath is interwoven by long N- and C-terminal extension arms emanating from each subunit, which create an extensive two-dimensional mesh that has the same connectivity in the extended and contracted state of the sheath. We propose that the contraction process draws energy from electrostatic and shape complementarities to insert the inner tube through bacterial cell membranes to eventually kill the bacteria.

Figure 1

Overall structure of precontraction pyocin R2. (a,b) Electron microscopy images of the pyocin R2 embedded in uranyl acetate stain (a) or vitreous ice (b). One precontraction pyocin particle is boxed. (c) Surface view of a 3D montage reconstruction of the entire precontraction pyocin. (d–g) Surface views of the 3D reconstruction of pyocin trunk. Each view corresponds to the segmentation patterns illustrated at left. 3D models and density maps of the attachment helix of the sheath and β-sheet region of the tube are shown in Supplementary Videos 1–4.

Figure 2

Structure of the pyocin tube compared to other tubes. (a) Ribbon diagram of a pyocin tube-protein monomer. Additional ribbon diagram is in Supplementary Video 5. (b) Ribbon diagram of two hexamers (discs). (c,d) Cut-away views as ribbon (c) and electrostatic (d) diagrams showing the inner surface of the structure in b. Charge distribution (red, negative; blue, positive; white, neutral) is shown in d. (e) Superposition of tube-protein structures from pyocin, λ phage (PDB 2K4Q, 29% residues superimposed, r.m.s. deviation 4.96 Å) and T6SS (Hcp, PDB 3EAA, 39% residues superimposed, r.m.s. deviation 3.32 Å). (f–h) As in d, but for T6SS (f), PS17 phage (g) and λ phage (h).

Figure 3

Structure of the pyocin sheath. (a,b) Side (a) and top (b) views of the atomic model of the pyocin trunk. Sheath subunits are shown in colors, and the tube is shown in gray. (c) Ribbon structure of the sheath monomer. Additional ribbon diagram is in Supplementary Video 6. (d) Topological diagram of the sheath monomer. (e) Joining of three (with numbers matching those in a) of the four adjacent monomers of the sheath protein, via β-sheet augmentation in their C domains (oval and inset). The polarities and identities of the β-strands in this augmented sheet are illustrated on the right.

Figure 4

Schematic diagram for the pyocin sheath topology of the extended mesh created by the N- and C-terminal extension arms within the sheath in the pre- and postcontraction states. β-strands participating in the sheet augmentation of the C domain are shown. α-helices involved in intersubunit interactions are shown as rectangles. Residue numbers for the subunit in red are given for strategic locations.

Figure 5

Sheath-tube interactions. (a,b) Side views of the interface between a sheath and a tube-protein subunit in ribbon diagram (a) and charged surface (b). (c) An open-book view of b. The complementary patches of interacting charges on both sheath and tube are marked with triangles.

Figure 6

Contraction of pyocin. (a,b) Comparison between the precontraction (a) and postcontraction (b) sheath. The cryo-EM density map in a is filtered to a resolution comparable to that in b (3.9 Å). Animation of contracted sheath is in Supplementary Video 7. (c,d) Ribbon diagrams of seven adjacent sheath subunits in their precontraction (c) and postcontraction (d) states. Morphing between c and d is shown in Supplementary Videos 8 and 9. (e) Overall rotational and translational movement of a single sheath monomer during contraction, illustrated in side and top views. The precontraction (left) and postcontraction (middle) states of a monomer are superimposed at right (blue, precontraction state; beige, postcontraction state).