Structural changes in bacteriophage T7 upon receptor-induced genome ejection

Abstract

Many tailed bacteriophages assemble ejection proteins and a portal–tail complex at a unique vertex of the capsid. The ejection proteins form a transenvelope channel extending the portal–tail channel for the delivery of genomic DNA in cell infection. Here, we report the structure of the mature bacteriophage T7, including the ejection proteins, as well as the structures of the full and empty T7 particles in complex with their cell receptor lipopolysaccharide. Our near–atomic-resolution reconstruction shows that the ejection proteins in the mature T7 assemble into a core, which comprises a fourfold gene product 16 (gp16) ring, an eightfold gp15 ring, and a putative eightfold gp14 ring. The gp15 and gp16 are mainly composed of helix bundles, and gp16 harbors a lytic transglycosylase domain for degrading the bacterial peptidoglycan layer. When interacting with the lipopolysaccharide, the T7 tail nozzle opens. Six copies of gp14 anchor to the tail nozzle, extending the nozzle across the lipopolysaccharide lipid bilayer. The structures of gp15 and gp16 in the mature T7 suggest that they should undergo remarkable conformational changes to form the transenvelope channel. Hydrophobic α-helices were observed in gp16 but not in gp15, suggesting that gp15 forms the channel in the hydrophilic periplasm and gp16 forms the channel in the cytoplasmic membrane.

Viral Ejection Proteins: Mosaically Conserved, Conformational Gymnasts

“…Four subunits related by four-fold rotational symmetry are visible in the procapsid [30] and maturevirion [28,29,31] reconstructions, although more subunits that do not obey strict rotational symmetry could be present inside the head and invisible to cryo-EM analysis. Only the N-terminal portion of gp16, named gp16-N (residues 1-228), was detected at high resolution in the post-ejection conformation (Figure 1B) [55], whereas gp16-C was visualized at low resolution in the lipid nanodiscs [55] and by cryo-ET [36], preventing direct comparison of the pre-ejection state that was visualized in situ in the mature T7 capsid [31]. We used all of the structural and biochemical information available in the literature to generate a composite model of the full-length gp16 in the post-ejection conformation (Figure 3C).…”

“…Recent in vitro studies have characterized the composition of the T7 ejection proteins and elucidated the high-resolution cryo-EM structures of gp15 and an N-terminal portion of gp16 (gp16-N) [ 55 , 56 ], which form a hexameric tunnel wide enough for dsDNA to pass through, bridging the outer membrane (OM) with the inner membrane (IM). In parallel, a recent high-resolution structure of the mature T7 virion [ 31 ] elucidated the atomic structures of the T7 ejection proteins in situ, arranged onto the portal protein. It became clear that T7 ejection proteins exist in two structurally distinct states: a pre-ejection conformation, in which they are coaxially arranged as rings on the portal to form a ‘core stack’ [ 28 , 29 , 30 , 31 , 32 , 36 ], and a post-ejection conformation, assembled as a transenvelope channel in the host-cell envelope called the DNA ejectosome [ 36 , 55 , 56 , 57 ].…”

“…In parallel, a recent high-resolution structure of the mature T7 virion [ 31 ] elucidated the atomic structures of the T7 ejection proteins in situ, arranged onto the portal protein. It became clear that T7 ejection proteins exist in two structurally distinct states: a pre-ejection conformation, in which they are coaxially arranged as rings on the portal to form a ‘core stack’ [ 28 , 29 , 30 , 31 , 32 , 36 ], and a post-ejection conformation, assembled as a transenvelope channel in the host-cell envelope called the DNA ejectosome [ 36 , 55 , 56 , 57 ].…”

“…Gp14 forms a pore in the OM: Gp14, the smallest ejection protein (196 residues), is entirely water-insoluble in vitro, requiring detergents to be extracted from the expression host’s membranes [ 47 , 58 ]. In the pre-ejection conformation, gp14 is solubilized by gp15 as part of the core stack [ 31 ] ( Figure 1 A). The N-terminus of gp14 (residues 1–78) was visualized by cryo-EM inside gp15’s dome-like structure, but due to the peculiar ring-like arrangement of gp14 and gp15 and the eight-fold symmetry used in the reconstruction, it was not possible to conclusively assign a copy number for gp14 that could be present in 8–20 copies [ 28 , 29 , 30 ].…”

“…The position of ejection proteins in the capsid has been a subject of intense investigation. These proteins can be dispersed throughout the capsid [ 12 ], exist in ordered structures above the portal protein [ 28 , 29 , 30 , 31 , 32 ] or loosely connected to the protein barrel [ 33 ], or even reside inside the tail complex [ 34 ]. Upon infection, ejection proteins are expelled inside the host, before the phage DNA [ 35 ], where they assemble a transient channel in the host-cell-envelope virion [ 34 , 36 , 37 ].…”