Cryo-EM structure of a Shigella podophage reveals a hybrid tail and novel decoration proteins

Abstract

There is a paucity of high-resolution structures of phages infecting Shigella, a human pathogen and a serious threat to global health. HRP29 is a Shigella podophage belonging to the Autographivirinae family, and has very low sequence identity to other known phages. Here, we resolved the structure of the entire HRP29 virion by cryo-EM. Phage HRP29 has a highly unusual tail that is a fusion of a T7-like tail tube and P22-like tailspikes mediated by interactions from a novel tailspike adaptor protein. Understanding phage tail structures is critical as they mediate hosts interactions. Furthermore, we show that the HRP29 capsid is stabilized by two novel, and essential decoration proteins, gp47 and gp48. Only one high resolution structure is currently available for Shigella podophages. The presence of a hybrid tail and an adapter protein suggests that it may be a product of horizontal gene transfer, and may be prevalent in other phages.

Keywords: bacteriophage; capsid; cryo-EM; virion; virus.

Figure 1:. HRP29 has a hybrid tail.

(A) Asymmetric reconstruction of HRP29 (EMD – 28227) with the central tail tube colored in blue and the tailspikes colored in orange (4.1 Å resolution). (B) Symmetry mismatch reconstruction of T7 (EMD – 31315) with the central tail tube colored in blue (7.0 Å resolution). (C) Asymmetric reconstruction of Sf6 (EMD – 5730) with the tail colored in orange highlighting the tailspike and tail needle (16.0 Å resolution). (D-F) Enlarged views of the tails of HRP29, T7 and Sf6.

Figure 2:. HRP29 has a central tail tube similar to T7

(A) Model of the tail generated from the focused C6 reconstruction of the tail with the portal gp35 colored in brown, adaptor gp39 in orange, nozzle gp40 in blue and tailspike adaptor gp44 in pink. (B-D) Side views of the tail tube proteins gp35, gp39 and gp40. (E) Top view of the tailspike adaptor gp44. (F) Interaction between gp35 (brown) and gp39 (orange) mediated by negatively charged amino acids on gp35 and positively charged amino acids on gp39. This interaction is flipped in terms of charge when compared to T7. (G) Negatively charged amino acids from two gp39 monomers interact with one monomer of gp40 at two distinct sites with positively charged amino acids. (H) Interaction between nozzle gp40 and tailspike adaptor gp44 mediated by Arg645 in gp40 and Asp25 in gp44. This interaction is also flipped in terms of charge when compared to T7.

Figure 3:. gp52 interacts with the tail through gp44.

(A) Representative 2D class averages of negatively stained gp44-gp52 complex purified from E. coli, shows a strong signal for tailspike protein gp52, whereas the signal for gp44 is more diffuse and noisy. (B) 3D reconstruction of gp44 and gp52 complex from a total of 8,583 particles show that the complex has an elbow shape. (C) Alphafold prediction of gp52 trimer (green) docked into the 3D reconstruction of gp44-gp52 complex; the alphafold prediction of gp44 trimer could not be reliably fit into the model.

Figure 4:. HRP29 mature virion has two decoration proteins gp47 and gp48.

(A) Model of HRP29 capsid generated form an icosahedral reconstruction with the capsid protein (gp37) colored in blue and the decoration protein gp47 colored in purple and magenta and the other decoration protein gp48 colored in yellow and green. (B) One of the monomers from the hexamers of the asymmetric unit from HRP29 and T7 were aligned against each other with a RMSD of 1.299 Å using match maker in chimera. (C) Monomer of gp47 from the penton-hexon junction. (D) Dimer of gp48 with the asterisk mark representing the two C-terminus. (E) gp47 interaction with the capsid proteins is stabilized by positively charged amino acid Arg63 from two capsid monomers. (F) gp48 interaction with the capsid is stabilized by a series of electrostatic interactions from each of the gp48 monomer mediated by residues Arg16, Lys23 and Asp34 from gp48 and Asp21, Asp23 and Lys139 of the capsid protein.

Figure 5:. CRISPR knockdown of gp44, gp47, gp48, gp52 and thermal stability assay.

(A) Spot assays of HRP29 and Sf6 at various dilutions on PE577, cells expressing a dead cas9 nuclease along with a gRNA targeting either gp44, gp47, gp48 or gp52. (B) Quantitative plaque assays of Sf6, HRP29 and T7 after incubation for 10 minutes at the indicated temperature. The efficiency of plating was determined as the titer of phage at the experimental temperature, divided by the titer of phage at the permissive temperature of 45 °C. Error bars reflect the standard deviation from at least three biological replicates.

Figure 6:. HRP29 procapsid has scaffolding protein density just beneath the N-terminus.

(A) Icosahedral model of the procapsid show a characteristic bumpy surface along with holes in the center of the pentons and hexons. (B) Interior view of the procapsid with the capsid density colored in grey and the scaffolding density from the difference map colored in magenta (resolution = 3.5 Å at FSC_0.143). The difference map was calculated by subtracting a map generated from the icosahedral model using the molmap command in chimera from the original cryo-EM density map. (C) Fit of scaffolding helix turn helix region shown in mesh representation and good fit was observed for the bulky amino acids. (D) The helix turn helix region of the C-terminal part of the scaffolding protein sits right under the modeled N-terminus which lacks the first 27 amino acids and the spine helix. The capsid protein is in blue and the scaffolding protein is colored in magenta.