Research ArticleSTRUCTURAL BIOLOGY

Structure and genome ejection mechanism of Staphylococcus aureus phage P68

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Science Advances  16 Oct 2019:
Vol. 5, no. 10, eaaw7414
DOI: 10.1126/sciadv.aaw7414
  • Fig. 1 Virion and genome organization of phage P68.

    (A and B) Structures of P68 virion, (C) genome release intermediate, and (D) empty particle. The whole P68 virion is shown in (A), whereas particles without the front half are shown in (B) to (D). The structures are colored to distinguish individual types of structural proteins and DNA. (E) Schematic diagram of P68 genome organization, with structural proteins color-coded in accordance with the structure diagrams shown in (A) to (D).

  • Fig. 2 Capsid structure of P68.

    (A) Major capsid proteins of P68 have HK97 fold and form T = 4 icosahedral lattice. Positions of selected icosahedral five-, three-, and twofold symmetry axes are indicated by pentagon, triangles, and oval, respectively. Borders of one icosahedral asymmetric unit are highlighted. (B) Cartoon representation of P68 major capsid proteins in icosahedral asymmetric unit. Positions of icosahedral symmetry axes and borders of icosahedral asymmetric unit are shown. (C) Major capsid proteins from icosahedral asymmetric unit differ in positions of elongated loops and N-terminal domains. Color coding of one of the subunits indicates division of major capsid protein to domains. (D) Residues 253 to 263 from the axial domain of major capsid proteins differ in structure. The residues form an α helix in the subunit that is part of the pentamers, whereas they constitute loops in the other subunits. The color coding of subunits is the same as in (B). (E) The inner capsid protein is organized in a T = 4 icosahedral lattice. Proteins are rainbow-colored from N terminus in blue to C terminus in red. Subunits related by icosahedral threefold axes and quasi-threefold axes of the T = 4 lattice form three-pointed stars in which the C terminus of one subunit is positioned next to the N terminus of another subunit. Borders of a selected icosahedral asymmetric unit are shown. (F) The ordering of the packaged P68 dsDNA genome (shown in blue) is disrupted around the fivefold vertices of the capsid (shown in yellow). Red triangle indicates one face of icosahedron. (G) Stacking interactions of two nucleotides with side chain of Trp74 of major capsid protein located next to fivefold vertex. (H) Side chains of Trp74 of major capsid proteins that form hexamers bind to His50 of inner capsid proteins.

  • Fig. 3 Structure of P68 portal complex and its interaction with capsid.

    (A) Twelvefold symmetrized structure of portal and inner core complexes of native P68. One of the portal proteins is colored according to domains: clip domain in magenta, wing in green, and stem in blue. Six inner core proteins associated with one portal protein subunit are highlighted in red. The inset shows the symmetry of the arrangement of the six inner core proteins. (B) Division of portal protein into domains. Color coding is the same as in (A). (C) Asymmetric reconstruction of portal complex showing interactions of one of the portal proteins highlighted in red, with DNA shown in blue. The interaction is indicated with a red arrow. One of the portal protein subunits that does not interact with the DNA is highlighted in green. (D) Detail of interaction of helix α9 of portal protein with DNA. Cryo-EM density is shown as gray transparent surface. (E) Structure of portal protein subunit that does not interact with DNA. (F) Interface between portal complex and capsid. Portal proteins are shown in gray, capsid proteins are shown in blue, and N termini of capsid proteins that mediate interactions with the portal are shown in pink and highlighted with pink arrows. (G) Side view of capsid-portal interactions. The single major capsid protein is shown in blue and its N terminus in pink, portal proteins in gray, inner core proteins in red, tail fibers in orange, and lower collar proteins in green. The inset shows detail of interactions between the N-terminal arm of the major capsid protein and stem domains of portal proteins.

  • Fig. 4 Head of P68 is decorated with five head fibers attached to hexamers of major capsid proteins located next to tail vertex.

    (A) P68 head is decorated with five head fibers that extend toward tail fibers. The head fibers can be divided into the N-terminal connector shown in orange, stalk in green, and receptor binding domain in pink. Because of the mismatch of the 5-fold symmetry of the head and 12-fold symmetry of the tail, only fibers 1, 2, and 4 are stabilized by interactions with tail fibers. (B to D) N-terminal connector domains of head fibers (shown in cartoon representation in orange) are attached to hexamers of major capsid proteins (shown as blue density). Cryo-EM density of inner capsid proteins is shown in yellow, and arms of inner core proteins, which interact with major capsid proteins, are shown in cartoon representation in red. Cryo-EM density of inner core proteins is shown as semitransparent red surface. External view of P68 head (B), section through capsid (C), and internal view of capsid (D). (E) Section through P68 head perpendicular to tail axis at level of inner core complex. Inner core proteins that interact with major capsid proteins are highlighted in red. The electron density of the inner core proteins is shown as a red semitransparent surface. (F to H) Details of organization of Phe259 side chains around quasi-sixfold axis of hexamer of major capsid proteins. In hexamers that interact with inner core proteins, side chains (in blue) are organized with threefold symmetry in alternating up and down conformations (F and G). In hexamers of capsid proteins that do not interact with inner core proteins, Phe259 side chains are organized with twofold symmetry, with two side chains pointing into capsid and four out (F and H).

  • Fig. 5 Structure of P68 tail.

    (A) Tail fibers of P68 form skirt around tail tube. Tail fibers are shown in gold; however, individual subunits of two tail fibers are distinguished in red, blue, and orange. Portal proteins are shown in magenta, inner core proteins in dark gray, and lower collar proteins in light gray. The inset shows details of interactions of tail fibers with each other and their attachment to the portal complex. (B) Structure of P68 tail fiber trimer in cartoon representation and its division into domains. The structure of the platform and tower domains was solved by x-ray crystallography to a resolution of 2.0 Å and fitted into the cryo-EM map of the native P68 tail. The inset shows a cartoon representation of the platform domain of the P68 tail fiber rainbow-colored from N terminus in blue to C terminus in red. (C) Structure of lower collar complex with individual subunits distinguished by rainbow coloring. (D) Division of lower collar protein into domains. The curly ring domain is shown in red and blue, the barrel domain is shown in yellow, and the dashed line indicates the knob-binding loop with unknown structure. (E) Surface charge distribution in inner core, portal, and lower collar complex of native P68 particle. (F) Fit of structure of tail needle of phage P22 into 12-fold symmetrized reconstruction of native P68 tail. (G) Sixfold symmetrized reconstruction of P68 tail knob and tail spike complexes. The surface of the cryo-EM map is radially colored on the basis of the distance from the sixfold axis of the complex. (H) Distribution of electron density in central section of tail knob and tail spike complexes. (I) Fit of structure of tail knob of phage C1 into P68 reconstruction. (J) Structure of tail spike with imposed fivefold symmetry shows its chalice and foot domains.

  • Fig. 6 Mechanism of P68 genome delivery into S. aureus cell.

    (A) Diagram of P68 virion. (B) Mechanism of P68 genome delivery. P68 virion attaches to cell surface by head or tail fibers (1). This attachment allows enzymes from tail spike to cleave bacterial cell wall (2). This degradation of S. aureus cell wall enables P68 to bind with its tail axis perpendicular to cell surface (3). Further cell wall digestion allows tip of P68 tail to reach cytoplasmic membrane, which triggers release of inner core proteins and DNA (4). Inner core proteins form channel in membrane for ejection of phage DNA into bacterial cytoplasm (5).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaaw7414/DC1

    Fig. S1. Purified sample of P68 contains native virions, particles in process of genome release, and empty particles; attachment of P68 virions to S. aureus cell walls; and interactions of P68 virions with liposomes.

    Fig. S2. Resolution and interpretability of cryo-EM reconstructions.

    Fig. S3. Details of P68 head.

    Fig. S4. Incorporation of P68 portal complex into capsid and changes in the structure of P68 portal complex upon genome release.

    Fig. S5. Structures of P68 tail fiber and tail spike.

    Fig. S6. Schemes of cryo-EM reconstruction strategies.

    Table S1. Cryo-EM structure quality indicators.

    Table S2. List of P68 proteins.

    Table S3. HHpred searches for homologs of P68 tail fiber, head fiber, tail knob, and tail spike.

    Table S4. X-ray structure quality indicators.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Purified sample of P68 contains native virions, particles in process of genome release, and empty particles; attachment of P68 virions to S. aureus cell walls; and interactions of P68 virions with liposomes.
    • Fig. S2. Resolution and interpretability of cryo-EM reconstructions.
    • Fig. S3. Details of P68 head.
    • Fig. S4. Incorporation of P68 portal complex into capsid and changes in the structure of P68 portal complex upon genome release.
    • Fig. S5. Structures of P68 tail fiber and tail spike.
    • Fig. S6. Schemes of cryo-EM reconstruction strategies.
    • Table S1. Cryo-EM structure quality indicators.
    • Table S2. List of P68 proteins.
    • Table S3. HHpred searches for homologs of P68 tail fiber, head fiber, tail knob, and tail spike.
    • Table S4. X-ray structure quality indicators.

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