Small terminase couples viral DNA binding to genome-packaging ATPase activity

Abstract

Packaging of viral genomes into empty procapsids is powered by a large DNA-packaging motor. In most viruses, this machine is composed of a large (L) and a small (S) terminase subunit complexed with a dodecamer of portal protein. Here we describe the 1.75 Å crystal structure of the bacteriophage P22 S-terminase in a nonameric conformation. The structure presents a central channel ∼23 Å in diameter, sufficiently large to accommodate hydrated B-DNA. The last 23 residues of S-terminase are essential for binding to DNA and assembly to L-terminase. Upon binding to its own DNA, S-terminase functions as a specific activator of L-terminase ATPase activity. The DNA-dependent stimulation of ATPase activity thus rationalizes the exclusive specificity of genome-packaging motors for viral DNA in the crowd of host DNA, ensuring fidelity of packaging and avoiding wasteful ATP hydrolysis. This posits a model for DNA-dependent activation of genome-packaging motors of general interest in virology.

Figure 1. Quaternary structure of the nonameric S-terminase subunit of bacteriophage P22

Ribbon diagram of S-terminase in side (A) and top (B) views. The oligomer is colored by secondary structure elements with α-helices, β-strands and loops in red, yellow and green, respectively. The overall diameter of S-terminase is ~95 Å with an internal hollow channel ~23 Å. (C) Secondary structure and amino acid sequence of bacteriophage P22 S-terminase subunit. Dashed in gray is the DNA-binding domain spanning residues 140–162, which is proteolytically cleaved in the 1.75 Å structure used for high resolution refinement and is disordered in the 3.35 Å structure of fl-S-terminase (Figure S1B). The illustration was generated using STRIDE (Heinig and Frishman, 2004). (D) Ribbon diagram of S-terminase protomer colored as in (A). The side chains for the YQ-motif on the tip of S-terminase are shown as sticks.

Figure 2. Conservation of S-terminase in tailed bacteriophages

Oligomer and protomer structure of S-terminase subunits in Podoviridae P22 (A, B) and Sf6 (C,D) (pdb 3HEF); Siphoviridae SF6 (E,F) (pdb 3ZQQ) and λ (G,H) (pdb 1J9I); Myoviridae T4-like phage 44RR (I,J) (pdb 3TXQ). In all cases, the S-terminase is displayed from the top with α-helices represented as cylinders; only one protomer per oligomer is colored by secondary structure elements, while the other subunits are in gray. Numbering of secondary structure elements in panels (B, D, F, H, J) is relative to P22 S-terminase. See also Figure S2.

Figure 3. Single particle analysis of negatively stained fl-S-terminase reveals two structurally distinct populations

Class averages of fl-S-terminase reveal particles characterized by a `short-' (A) and an `extended-barrel' (B). Particles in (B) exhibit an extension of the DNA-channel by ~30Å as compared to particles in (A). A 3D-negative stain reconstruction (in gray) of S-terminase with a `short barrel' is overlaid to a ribbon model of the crystallographic structure of cl-S-terminase spanning residues 1–139, shown in top (C) and side (D) views. Top (E) and side (F) views of a 3D-negative stain reconstruction of S-terminase particles with an `extended barrel' are overlaid to a model of the fl-S-terminase that includes the crystallographic structure and an hypothetical model of C-terminal residues 140–158 produced by Modeller (Sali and Blundell, 1993) (Figure S3 F,G).

Figure 4. P22 S-terminase DNA-binding activity

EMSA of fl-S-terminase (A) or ΔC140-S-terminase (B) binding to gp3-DNA. In both panels, lanes 2–11 show a titration of 0- to 24-fold equivalents of fl- and ΔC140-S-terminase incubated with the gp3-DNA and separated on a 1.5% agarose gel followed by ethidium bromide staining. (C) Quantification of EMSAs in (A,B) based on four independent repeats. The binding of fl-S-terminase (D) or ΔC140-S-terminase (F) to L-terminase was characterized by SEC on a Superose 12 column; elution peaks for S-terminase, L-terminase and the S/L-terminase complex are shown in green, blue and red, respectively. (E) and (G) show SDS-analysis of fractions eluted from gel filtration in panel (D) and (F), respectively.

Figure 5. S-terminase activates the ATPase activity of L-terminase in the presence of gp3-DNA

(A) ATPase assay resolved on PEI-TLC in the presence of different reactants and γ³²-ATP. The position of γ³²-ATP and ³²P_i is indicated by arrows (B). Quantification of ³²P_i released during the ATPase assay. The intensity of ³²P_i released by L-terminase in the presence of S-terminase and gp3-DNA (lanes 4–5 in Figure 5A) was corrected by subtracting the intensity of ³²P_i released in control reactions containing only S-terminase, with and without gp3-DNA (lanes 8–9 in Figure 5A). Error bars are calculated from averaging the intensity of ³²P_i released over five independent experiments carried out under identical conditions. The average standard deviation is usually less than 3%.