Assembly of bacteriophage P2 capsids from capsid protein fused to internal scaffolding protein

Abstract

Most tailed bacteriophages with double-stranded DNA genomes code for a scaffolding protein, which is required for capsid assembly, but is removed during capsid maturation and DNA packaging. The gpO scaffolding protein of bacteriophage P2 also doubles as a maturation protease, while the scaffolding activity is confined to a 90 residue C-terminal "scaffolding" domain. Bacteriophage HK97 lacks a separate scaffolding protein; instead, an N-terminal "delta" domain in the capsid protein appears to serve an analogous role. We asked whether the C-terminal scaffolding domain of gpO could work as a delta domain when fused to the gpN capsid protein. Varying lengths of C-terminal sequences from gpO were fused to the N-terminus of gpN and expressed in E. coli. The presence of just the 41 C-terminal residues of gpO increased the fidelity of assembly and promoted the formation of closed shells, but the shells formed were predominantly small, 40 nm shells, compared to the normal, 55 nm P2 procapsid shells. Larger scaffolding domains fused to gpN caused the formation of shells of varying size and shape. The results suggest that while fusing the scaffolding protein to the capsid protein assists in shell closure, it also restricts the conformational variability of the capsid protein.

Keywords: Assembly; Cryo-electron microscopy; Procapsid; Size determination; Virus.

Fig. 1

gpO and gpN co-expression and fusion protein constructs. Predicted α-helices are indicated as squiggly lines on top of the gpO protein. The 357 residue gpN protein is shown cross-hatched and is not drawn to scale, as indicated by the jagged line. The gpO truncations start at the indicated residues (142, 195, 244, or 261) and end at the gpO C-terminus (residue 284). All constructs include the full-length gpN protein

Fig. 2

Schematic diagram comparing the order of the protease (open box), scaffolding (cross hatched) and capsid (dotted) activities in the genomes of HK97 and P2 (drawn to scale). In HK97, the gene encoding the protease (gp4) precedes the gene for the capsid protein (gp5), which incorporates an N-terminal scaffolding domain. In P2, the protease and scaffolding activities are both contained within the gpO protein. The last line represents any of the various constructs that were made by fusing the C-terminal scaffolding domain of O with the N capsid gene

Fig. 3

Size distribution histogram of the particles formed by gpN alone and the various co-expression and fusion constructs described in the text. Open bars represent aberrant particles; solid bars, P4-like, small particles (<46 nm); and hatched bars, P2-like, large particles (≥46 nm). The number of particles counted for each construct (n) is shown above the bars

Fig. 4

Cryo-EM of structures produced by expression of co-expression clones O(141–284) + N (A), O(195–284) + N (B) and O(244–284) + N (C); fusion protein clones O(141–284)::N (D), O(195– 284)::N (E) and O(244–284)::N (F); and fusion/co-expression clones O(141–284)::N + N (G) and O(195–284)::N + N (H) and O(244–284)::N + N (I). Examples of well-formed particles of P2 and P4 size are indicated in each panel (where available) by black (P2) and white (P4) arrows. Some of the many thin-walled shells produced in (D) are indicated with asterisks. Scale bar, 100 nm

Fig. 5

a Coomassie-stained SDS–PAGE of proteins expressed by fusion protein clones. Lane 1, O(142–284)::N; Lane 2, O(195–284)::N; lane 3, O(244–284)::N. b SDS–PAGE of gpN alone (lane 1), the O(195–284)::N fusion protein (lane 2), and the proteins expressed from the O(195–284)::N + N co-expression clone (lane 3). M marker, MW indicated (kDa)

Fig. 6

Radial profiles of fusion protein capsids. Top row, two-dimensional averages of a 71 P4 procapsids from a gpN + Sid coexpression, b the O(195–284)::N fusion protein (69 particles), and (c) the O(195–284)::N + N fusion co-expression clone (71 particles). Bottom row (d–f), radial averages of the same three sets of images. The graph (g) shows the radially averaged density as a function of radius (nm) for the gpN + Sid co-expression (solid black line), O(195–284)::N fusion (gray line) and O(195– 284)::N + N (dashed black line). The density maxima corresponding to gpN, gpO, and Sid are indicated on the graph

Fig. 7

Model for gpO–gpN interaction. The “L” shapes represent the gpN monomers. The N-terminal arm is indicated. a shows the interaction between O(195–284) and gpN in an O(195–284) + N co-expression experiment. The C-terminal Cys284 residues of gpO are thought to interact transiently with gpN, while the α-helical regions (residues 195–243, shown as cylinders) dimerize to promote shell assembly. b and c show the O(195–284) and O(244–284) fragments of gpO, respectively, covalently fused to the N-terminus of gpN in the O(195–284)::N and O(244–284)::N fusion clones