Atomic structure of bacteriophage Sf6 tail needle knob - PubMed (original) (raw)

Atomic structure of bacteriophage Sf6 tail needle knob

Anshul Bhardwaj et al. J Biol Chem. 2011.

Abstract

Podoviridae are double-stranded DNA bacteriophages that use short, non-contractile tails to adsorb to the host cell surface. Within the tail apparatus of P22-like phages, a dedicated fiber known as the "tail needle" likely functions as a cell envelope-penetrating device to promote ejection of viral DNA inside the host. In Sf6, a P22-like phage that infects Shigella flexneri, the tail needle presents a C-terminal globular knob. This knob, absent in phage P22 but shared in other members of the P22-like genus, represents the outermost exposed tip of the virion that contacts the host cell surface. Here, we report a crystal structure of the Sf6 tail needle knob determined at 1.0 Å resolution. The structure reveals a trimeric globular domain of the TNF fold structurally superimposable with that of the tail-less phage PRD1 spike protein P5 and the adenovirus knob, domains that in both viruses function in receptor binding. However, P22-like phages are not known to utilize a protein receptor and are thought to directly penetrate the host surface. At 1.0 Å resolution, we identified three equivalents of l-glutamic acid (l-Glu) bound to each subunit interface. Although intimately bound to the protein, l-Glu does not increase the structural stability of the trimer nor it affects its ability to self-trimerize in vitro. In analogy to P22 gp26, we suggest the tail needle of phage Sf6 is ejected through the bacterial cell envelope during infection and its C-terminal knob is threaded through peptidoglycan pores formed by glycan strands.

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Figures

FIGURE 1.

FIGURE 1.

Bacteriophage Sf6 tail needle forms an elongated fiber with a C-terminal globular domain. A, micrographs of negatively stained Sf6 tail needle fibers. B, five class averages resulting from several iterations of multireference alignment (24). The approximate length of the Sf6 tail needle estimated from the projection average is ∼220 Å. C, schematic diagram of the Sf6 tail needle (residues 1–282) and the knob construct used in this study (residues 132–282).

FIGURE 2.

FIGURE 2.

Atomic structure of bacteriophage Sf6 tail needle knob bound to l-Glu visualized crystallographically at 1.0 Å resolution. A and B, ribbon diagram of the trimeric Sf6 tail needle knob shown in side and top views, respectively. The structure is colored by secondary structure elements with β-strands and α-helices in yellow and red, respectively.

l

-Glu and phosphate ion are shown as ball-and-stick diagrams. C, ribbon diagram of the tail needle knob monomer reveals the characteristic architecture of a TNF jellyroll fold consisting of eight β-strands organized in two β-sheets with a tilt angle of 36°.

FIGURE 3.

FIGURE 3.

l-Glu binding cleft in Sf6 tail needle knob. A, surface representation of Sf6 knob highlighting the interface between subunits A and B (colored in blue and red, respectively). B, zoom-in view of the subunit interface where

l

-Glu is bound. Subunit A and B side chains from the final refined model are shown in sticks, and

l

-Glu is shown in a stick-and-ball representation. C, the final refined model for

l

-Glu and the interacting residues from both subunits are overlaid to the final 2_Fo_ − Fc electron density map calculated at 1.0 Å resolution and contoured at 1.5σ above background (in blue). The

l

-Glu is bound in a cleft formed at the interface of two knob monomers, closely surrounded by B-Glu146, B-Lys277, A-Leu255, B-Lys200, A-Ser248, and A-Asp250. D, thermal stability of the native and GdnHCl-treated Sf6 knob. Both samples unfold irreversibly with a melting temperature (Tm) of ∼67 °C. E, SDS resistance assay of wild type Sf6 tail needle and Sf6 knob that were previously treated under native conditions (N) or dialyzed against 1.5

m

GdnHCl, 0.5

m

NaCl (D). RT and 95° denote samples that were either incubated at 22 °C or boiled for 5 min prior to being electrophoresed.

FIGURE 4.

FIGURE 4.

Comparison of the Sf6 tail needle knob with the receptor-binding domains of PRD1 and adenovirus. Left panel, Sf6 knob (A); PRD1 P5 (PDB code 1YQ5) (B); and adenovirus knob (PDB code 1NOB) (C). Right panel, topological diagrams of each protomer (generated using PDBsum (50)). Red and yellow are α-helices and β-strands, respectively.

FIGURE 5.

FIGURE 5.

Modeling bacteriophage tail needles. A, crystal structure of P22 tail needle gp26 (PDB code 3C9I). B, homology model of the full-length Sf6 tail needle. The portion of the tail needle helical core missing (residues 1–132) in the construct used for crystallography was modeled using Phyre (38) and is highlighted in gray. C, crystal structure of the receptor binding protein from lactococcal phage TP901-1 base plate (PDB code 3EJC) (20, 39). D, a composite model of bacteriophage P22 and Sf6 (E) mature virions generated by fitting the atomic structure of gp26 tail needles and tailspikes into the asymmetric cryo-EM reconstruction of mature P22 (EMDB ID 1220) (6).

FIGURE 6.

FIGURE 6.

A structural model for Sf6 tail needle penetration of Shigella cell wall. Top (A), tilted (B), and side (C) view of the Sf6 tail needle embedded into a pore of the Gram-negative cell wall, according to the structure of PG determined by Meroueh et al. (49). Glycan strands are colored orange, and the cross-linking stem peptides are colored green, with

d

-Glu in purple. The Sf6 tail needle is shown as a transparent solvent-accessible gray surface overlaid to the ribbon structure.

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