Structural Plasticity of the Protein Plug That Traps Newly Packaged Genomes in Podoviridae Virions

Abstract

Bacterial viruses of the P22-like family encode a specialized tail needle essential for genome stabilization after DNA packaging and implicated in Gram-negative cell envelope penetration. The atomic structure of P22 tail needle (gp26) crystallized at acidic pH reveals a slender fiber containing an N-terminal "trimer of hairpins" tip. Although the length and composition of tail needles vary significantly in Podoviridae, unexpectedly, the amino acid sequence of the N-terminal tip is exceptionally conserved in more than 200 genomes of P22-like phages and prophages. In this paper, we used x-ray crystallography and EM to investigate the neutral pH structure of three tail needles from bacteriophage P22, HK620, and Sf6. In all cases, we found that the N-terminal tip is poorly structured, in stark contrast to the compact trimer of hairpins seen in gp26 crystallized at acidic pH. Hydrogen-deuterium exchange mass spectrometry, limited proteolysis, circular dichroism spectroscopy, and gel filtration chromatography revealed that the N-terminal tip is highly dynamic in solution and unlikely to adopt a stable trimeric conformation at physiological pH. This is supported by the cryo-EM reconstruction of P22 mature virion tail, where the density of gp26 N-terminal tip is incompatible with a trimer of hairpins. We propose the tail needle N-terminal tip exists in two conformations: a pre-ejection extended conformation, which seals the portal vertex after genome packaging, and a postejection trimer of hairpins, which forms upon its release from the virion. The conformational plasticity of the tail needle N-terminal tip is built in the amino acid sequence, explaining its extraordinary conservation in nature.Podoviridae forms a family of bacterial viruses (bacteriophages) characterized by short and noncontractile tails (1). The tail complex is a sophisticated molecular machine, which is attached to a unique vertex of the icosahedral capsid and provides an entry through which the viral genome is packaged during replication and is ejected into the host during infection (2). In the prototypical Salmonella enterica phage P22 (3), the tail machine consists of a ϳ2.8-MDa multisubunit complex (4, 5) that replaces a single penton of the icosahedral capsid (6 -8). P22 binds to the Salmonella surface via tailspike proteins (gp9) that provide adsorption specificity by binding to the Salmonella O-antigen surface polysaccharide and by cleaving it (9). In addition to the tailspike, P22 virions contain a tail needle that is located at the distal tip of the tail axis (5-7), projecting ϳ140 Å outwards from the virion. In P22, the tail needle protein is encoded by gene 26 (10, 11) and at acid pH forms a 240-Å-long trimeric coiled-coil fiber containing three domains: an N-terminal tip (NTT), 3 a central ␣-helical coiled coil core, and a C-terminal tip (CTT) that folds as an inverted coiled-coil (12-14). Orthologues of gene 26 have been identified bioinformatically in hundreds of phage genomes and prophages (15). Significant sequence variability exists ...

Real-space refinement inPHENIXfor cryo-EM and crystallography

“…This is a work in progress, and further details and advances will be reported as the program evolves. To date, phenix.real_space_refine has been used in a number of documented structural studies (see, for example, Fischer et al, 2015;Shalev-Benami et al, 2016;Chua et al, 2016;Ahmed et al, 2016;Yang et al, 2016;Gao et al, 2016;Chen et al, 2016;Bhardwaj et al, 2016;Lokareddy et al, 2017;Hryc et al, 2017;Ahmed et al, 2017;Demo et al, 2017;Paulino et al, 2017;Liu et al, 2017). Fig.…”

Cryo-EM Elucidation of the Structure of Bacteriophage P22 Virions after Genome Release

“…One hypothesis is that the peptide linker between the tailspike protein's particle (capsid) binding domain and its receptor binding domain relays the signal of phage-receptor binding sensed by the tailspike (39). Another hypothesis is that penetration of the gp26 needle into the outer membrane triggers the release of the ejection proteins and genome (40). The C-terminal domain of the gp26 needle plugs the phage chromosome's exit channel (40,41), and in gp26 deletion mutants, P22 virion assembly and DNA packaging occur normally, but the packaged DNA is unstable and spontaneously leaks out of these particles (41,42).…”

“…Another hypothesis is that penetration of the gp26 needle into the outer membrane triggers the release of the ejection proteins and genome (40). The C-terminal domain of the gp26 needle plugs the phage chromosome's exit channel (40,41), and in gp26 deletion mutants, P22 virion assembly and DNA packaging occur normally, but the packaged DNA is unstable and spontaneously leaks out of these particles (41,42). The resulting empty particles-which lack gp26 and DNA and are sometimes called ''empty heads''-contain coat protein, portal protein, tailspike protein, and all three ejection proteins (13,43).…”

Kinetic Analysis of Bacteriophage Sf6 Binding to Outer Membrane Protein A Using Whole Virions

“…Upon interaction with Omps by the tail machinery, a conformational change in the phage is likely triggered. Amino acid substitutions in the loops of OmpA may affect the ability of the phage to adopt the correct conformation to promote channel formation, which is necessary to translocate the DNA genome [44,[49][50][51]. Although more work is necessary to discern a complete understanding of Sf6 (and Podoviridae) infection, the data presented here shed light on the kinetics of Sf6 and OmpA binding, which is an important step during host recognition.…”