Structure and size determination of bacteriophage P2 and P4 procapsids: function of size responsiveness mutations

Abstract

Bacteriophage P4 is dependent on structural proteins supplied by a helper phage, P2, to assemble infectious virions. Bacteriophage P2 normally forms an icosahedral capsid with T=7 symmetry from the gpN capsid protein, the gpO scaffolding protein and the gpQ portal protein. In the presence of P4, however, the same structural proteins are assembled into a smaller capsid with T=4 symmetry. This size determination is effected by the P4-encoded protein Sid, which forms an external scaffold around the small P4 procapsids. Size responsiveness (sir) mutants in gpN fail to assemble small capsids even in the presence of Sid. We have produced large and small procapsids by co-expression of gpN with gpO and Sid, respectively, and applied cryo-electron microscopy and three-dimensional reconstruction methods to visualize these procapsids. gpN has an HK97-like fold and interacts with Sid in an exposed loop where the sir mutations are clustered. The T=7 lattice of P2 has dextro handedness, unlike the laevo lattices of other phages with this fold observed so far.

Figure 1

Schematic diagram of the P2/P4 capsid assembly pathway. (A) Capsid protein gpN, scaffolding protein gpO and connector (portal) protein gpQ are assembled into the T=7 P2 procapsid. (B) GpN, gpO and gpQ are then processed to their mature forms, N*, O* and Q*, respectively. DNA is packaged into the procapsid by the P2 terminase complex (gpM and gpP), accompanied by expansion into a mature capsid (B). (C) In the presence of the P4-encoded protein Sid, P2 structural proteins are instead assembled into a small, T=4 procapsid with Sid forming an external scaffold. (D) Loss of Sid, (E) protein processing and DNA packaging leads to the mature, expanded P4 capsid. (F) The decoration protein Psu is added to the mature capsid.

Figure 2

Cryo-electron micrographs of P4 (A) and P2 (B) procapsids. Scale bar, 100 nm.

Figure 3

Isosurface representations of the P4 (A) and P2 (B) procapsid reconstructions, viewed down a twofold axis of symmetry. The large triangles correspond to one icosahedral face, delimited by three fivefold axes of symmetry (pentagons). Three twofold symmetry axes (ovals) are located along the edges of the triangle. The four non-equivalent gpN subunits in the asymmetric unit in P4 and the seven subunits in P2 are labeled from A through G. The same triangle is represented schematically below the corresponding reconstructions, showing the subunit arrangement in the T=4 and T=7 dextro lattices. The icosahedral (type 1) threefold axis is indicated by a filled triangle, while the local type 2 and type 3 threefold axes are indicated by open triangles.

Figure 4

Structure of gpN and HK97 gp5. (A) Alignment of the secondary structure elements of gpN and gp5. α-helices and β-strands are shown as boxes and arrows, respectively, labeled according to (Helgstrand et al., 2003) and colored as follows: red, N-arm; pink, E loop; blue, P domain β sheet; gold, spine helix motif; green, A domain. The dashed lines in gp5 correspond to insertions in gpN. The purple triangles indicate the location of sir mutations in the gpN sequence. (B) Ribbon diagram of HK97 gp5, colored as in (A). (C) Ribbon diagram of HK97 gp5 fitted into the P2 procapsid (gpN subunit B) density, after adjusting the angle between the A and P domains (colored as in A and B). The original, unmoved gp5 is shown in gray. (D) Comparison of fitting the HK97 P domain into the P2 procapsid hexamer density in the T=7 laevo and T=7 dextro lattices. Three subunits are shown (gold, red, purple).

Figure 5

Modeling of gpN. (A) Ribbon diagram showing the superposition of the top two ITASSER models (red and blue) on the HK97 gp5 template (gray). (B) The gpN model, based on the I-TASSER models, modified to fit in the P2 procapsid map, colored the same way as gp5 in Fig. 4. Pertinent secondary structure elements are labeled. The amino acids affected by the sir mutations are shown as purple spheres. (C) The gpN model fitted into the P2 procapsid subunit B density. (D) gpN model fitted into the P4 procapsid subunit B density. The density corresponding to Sid is shown in red, with a model for the proposed C-terminal helix fitted in.

Figure 6

GpN and Sid interactions. (A) Isosurface representation (mesh) of a hexamer from the P2 procapsid reconstruction, viewed down the quasi-sixfold axis. The six gpN subunits are shown in ribbon representation (B, yellow; C, red; D, blue; E, purple; F, green; G, orange). (B) Oblique view of the same hexamer (yellow isosurface), showing details of the interactions between two subunits, B (yellow) and C (red). (C) View of the P4 hexamer, viewed down the icosahedral twofold axis, with gpN subunits fitted (B, yellow; C, red; D, blue). The Sid scaffold is shown as a solid red isosurface. Icosahedral threefold axes are indicated by triangles. (D) Oblique view of the P4 procapsid hexamer, including the Sid scaffold. (E) Isosurfaces of the P2 (blue mesh) and P4 (yellow solid surface) hexamers superimposed. The calculated difference map between the two is shown as a red isosurface, showing that the only significant difference between the P2 and P4 hexamers is the Sid scaffold. (F) Superposition of P2 (blue mesh) and P4 (red mesh) density at the type 2 (ABC) trivalent interaction. The three subunits involved in the interaction are shown in ribbon representation (A, pink; B, yellow; C, red).