Conformational changes in and translocation of small proteins: insights into the ejection mechanism of podophages

Abstract

Podophage tails are too short to span the cell envelope during infection. Consequently, podophages initially eject the core proteins within the head for the formation of an elongated trans-envelope channel for DNA ejection. Although the core proteins of bacteriophage T7 have been resolved at near-atomic resolution, the mechanisms of core proteins and DNA ejection remain to be fully elucidated. In this study, we provided improved structures of core proteins in mature T7 and the portal-tail complex in lipopolysaccharide-induced DNA-ejected T7 to resolutions of approximately 3 Å. Using these structures, we identified three small proteins, namely gp14, gp6.7, and gp7.3, and illustrated the conformational changes in and translocation of these proteins from the mature to DNA-ejected states. Our structures indicate that gp6.7, which participates in the assembly of the core and trans-envelope channel, is a core protein, and that gp7.3 serves as a structural scaffold to assist the assembly of the nozzle into the adaptor.

Importance: Podophage T7 core proteins form an elongated trans-envelope channel for genomic DNA delivery into the host cell. The structures of the core proteins within the mature T7 and assembled in the periplasmic tunnel form in the DNA-ejected T7 have been resolved previously. Here, we resolved the structures of two new structural proteins (gp6.7 and gp7.3) within mature T7 and receptor-induced DNA-ejected T7. The gp6.7 protein participates in the assembly of the core complex within mature T7 and the trans-envelope channel during T7 infection; therefore, gp6.7 is a core protein. Before T7 infection, gp7.3 plays a role in promoting the assembly of the nozzle into the adaptor.

Keywords: bacteriophages; core proteins; dsDNA ejection; trans-envelope channel.

Fig 1

Overall structures of mature and DNA-ejected T7. (A) Side (left) and cut-open (right) views of the entire asymmetric structure of mature T7. Color code applies to panels A–E. (B) Cut-open view of high-resolution density map of the core–portal–tail complex in mature T7. (C) Superposition of our atomic models (sticks) on density maps (mesh) extracted from box (portal) in panel B. (D) Side (left) and cut-open (right) views of the entire asymmetric structure of DNA-ejected T7. (E) Cut-open view of high-resolution density map of the portal–tail complex in DNA-ejected T7.

Fig 2

Structure of core–portal–tail complex in mature T7. (A) Side (left) and slab (right) views of atomic models (ribbons) of core–portal–tail complex in mature T7. Color coding is identical to that in Fig. 1B. Middle gray rod is a density map of DNA. (B) Cross-section view of columns in A showing interactions among portal and proteins gp6.7, gp14, and gp15. (C) Ribbon model (left) of gp14 molecule and zoomed-in view (right) of atomic model superimposed on its density map (mesh). (D) Zoomed-in view of interactions among proteins gp6.7, gp8, gp14, and gp15. (E, F) Atomic models (sticks) of gp6.7 loop I (residues 2–15, E) and loop II (residues 34–50, F) superimposed on their density maps (mesh). (G) Cross-section view of columns in A showing interactions between gp6.7 and gp15. (H) Top (upper) and cut-open (bottom) views of interactions between dsDNA and protein 7.3. Residues K84 and K85 of gp7.3 are labeled. (I) Atomic model (stick) of gp7.3 superimposed on its density map (mesh).

Fig 3

Structure of the core–portal–tail complex in DNA-ejected T7. (A) Side (center) and slab (right) views of atomic models (ribbons) of portal–tail complex in DNA-ejected T7. In the left inset, hairpin A from one gp12 molecule and hairpin B from adjacent gp12 molecule interact with the gp14–gp6.7 channel inside tail nozzle. Color coding is identical to that in Fig. 1D. (B) Side (upper) and bottom (bottom) views of atomic models (ribbon) of the gp14–gp6.7 channel. (C) Atomic model of gp14 superimposed on its density map (mesh). (D) Atomic models of conformers A and B of gp6.7 superimposed on their density maps (mesh). (E) Superposition of atomic models of conformers A and B of gp6.7 for showing conformational differences.

Fig 4

Conformational and location changes from mature to DNA-ejected states of three small proteins of gp14, gp6.7, and gp7.3. (A) Diagram of mature T7. (B) Diagram of DNA-ejected T7. (C, D) Slab (C) and cross-section (D) views of columns in A showing interactions among proteins gp6.7, gp14, and gp15, and portal. (E) Cross-section views of columns in A showing interactions among gp7.3, adaptor, and nozzle. (F) Cross-section views of columns in B showing interactions between gp6.7, gp14, and nozzle. Color coding is identical to that in Fig. 1.