Architecture of the flexible tail tube of bacteriophage SPP1

Abstract

Bacteriophage SPP1 is a double-stranded DNA virus of the Siphoviridae family that infects the bacterium Bacillus subtilis. This family of phages features a long, flexible, non-contractile tail that has been difficult to characterize structurally. Here, we present the atomic structure of the tail tube of phage SPP1. Our hybrid structure is based on the integration of structural restraints from solid-state nuclear magnetic resonance (NMR) and a density map from cryo-EM. We show that the tail tube protein gp17.1 organizes into hexameric rings that are stacked by flexible linker domains and, thus, form a hollow flexible tube with a negatively charged lumen suitable for the transport of DNA. Additionally, we assess the dynamics of the system by combining relaxation measurements with variances in density maps.

Fig. 1. Solid-state NMR data for hybrid structure calculation defines local structure.

a Backbone dihedral angles define the secondary structure (arrows represent β-sheets, barrels α-helices). The loop (40–59) and the C-arm (143–176) are highlighted in purple and pink, respectively. b Isoleucine Cδ1-methyl, alanine Cβ-methyl, leucine/valine Cδ/Cγ-methyl, threonine Cγ2-methyl, and methionine Cε-methyl labeling renders those moieties NMR-visible and yields highly resolved NMR spectra (green). c Long-range restraints between amide protons are extracted from a 4D HNhhNH spectrum (orange), whereas long-range restraints between amide and methyl groups are extracted from a series of 3D HNhH spectra (blue). 2D planes from both types of spectra are superimposed on a 2D hNH fingerprint spectrum (gray). d Representative long-range restraints (dashed lines) defining the inner β-barrel of the tail tube. The colored β-strands belong to one monomer, whereas the gray β-strands are from the neighboring subunits. Amide–amide contacts are highlighted in violet, amide–methyl contacts in cyan. e Schematic representation of long-range distance restraints between the Cδ1 methyl group of Ile18 and amide groups, defining the interface between the N-terminus of one monomer i (including Ile18; in cyan) and the C-arm (143–176) of another monomer j (in pink) within the tail tube. Protons are colored in red.

Fig. 2. Cryo-EM data for hybrid structure calculation provides global and local information.

a 3.5–6 Å cryo-EM map of the tail tube of SPP1 consisting of polymerized gp17.1 subunits does not only allow to deduce symmetry restraints but also limits the position of all atoms within the density. Rings with a thickness of 38.5 Å (as represented by the straight arrows) stack onto each other with a rotation of 21.9° (as represented by the bent arrow). b–d Local resolution of the cryo-EM map increases going from outer to the inner surface of the tail tube (as represented by the color gradient). e The inner region of the map reveals a highly resolved β-barrel and allows for the positioning of bulky sidechains as exemplified for Tyr67 and Tyr68 (f). The direction of the tail structure is baseplate upwards.

Fig. 3. Structure of polymerized gp17.1 forming the tail tube of the bacteriophage SPP1.

a Final ten lowest-energy structures of a gp17.1 subunit which consist of a central β-sandwich-type fold (turquoise) that is flanked by an α-helix (pink), a large loop and an extended C-terminal arm (C-arm). b Six gp17.1 monomers form a hexameric ring. The inner β-sheets of the β-sandwiches organize in a β-barrel motif that forms the lumen of the tube. c These hexameric rings stack onto each other in a helical fashion creating a hollow tube. Ring-to-ring contacts are mediated by the two loop regions (highlighted in red)—especially by the C-arm that folds onto the subjacent ring. d The molecular lipophilicity potential of gp17.1 reveals a hydrophobic patch on the surface of one subunit i. The color gradient represents the lipophilicity potential. e, f This unpolar area is obscured by the C-arm (pink) of the superjacent subunit j within the complex of the tail-tube—by anchoring the sidechain of Gln162 into a pocket. g The loop of subunit i (turquoise) features mostly electrostatic interactions with five neighboring subunits (gray, purple, green, beige, and orange) within the complex. Charged amino acids are colored in red (negative) and blue (positive). The direction of the tail structure is baseplate upwards.

Fig. 4. Bending of the tail tube is mediated by flexible hinge regions.

a Model of a bent SPP1 tail tube with a curvature radius of 655 Å (as indicated by the arrow). The model is based on the structure of straight tubes and 2D class averages of bent tubes. Most structural changes are found on the outside of the curve (red) which implies that the bending process is mediated by stretching. b Regions that act as hinges during bending of the tube are colored in pink. c Variances in the cryo-EM map (pink) match the hinge regions. d Also, hinge regions are associated with highest ¹⁵N R₁ (color key) and R_1ρ (thickness of wire) relaxation rates. White coloring represents missing values. e Decaying relaxation dispersion profiles indicate the presence of slow motions. f Relaxation dispersion profiles of the inner β-barrel can be fitted in a correlated manner to a two-state exchange process. Smallest chemical shift changes correlate with middle regions of the β-barrel which are furthest away from the hinge regions (color key). The direction of the tail structure is baseplate upwards. Source data are provided as a Source Data file.