'Let the phage do the work': using the phage P22 coat protein structures as a framework to understand its folding and assembly mutants

Abstract

The amino acid sequence of viral capsid proteins contains information about their folding, structure and self-assembly processes. While some viruses assemble from small preformed oligomers of coat proteins, other viruses such as phage P22 and herpesvirus assemble from monomeric proteins (Fuller and King, 1980; Newcomb et al., 1999). The subunit assembly process is strictly controlled through protein:protein interactions such that icosahedral structures are formed with specific symmetries, rather than aberrant structures. dsDNA viruses commonly assemble by first forming a precursor capsid that serves as a DNA packaging machine (Earnshaw, Hendrix, and King, 1980; Heymann et al., 2003). DNA packaging is accompanied by a conformational transition of the small precursor procapsid into a larger capsid for isometric viruses. Here we highlight the pseudo-atomic structures of phage P22 coat protein and rationalize several decades of data about P22 coat protein folding, assembly and maturation generated from a combination of genetics and biochemistry.

Figure 1. P22 assembly pathway including known structures

Shown in the top panel is the morphogenic pathway of phage P22. Below are the corresponding structures as follows: P22 coat protein in the procapsid state determined by cryo-EM homology modeling (PDB entry 3IYI) (Parent et al., 2010); Scaffolding protein C-terminal coat-protein binding domain NMR structure (PDB entry 2GP8) (Sun et al., 2000); Procapsid cryo-EM density map (Jiang et al., 2003); gp3 terminase cryo-EM density map (modified from Nemecek et all 2008, JMB) (Nemecek et al., 2008); Tail Needle (gp26 trimer) crystal structure (PDB entry 2POH) (Olia, Casjens, and Cingolani, 2007); Tailspike trimer crystal structure (PDB entry 1TSP) (Steinbacher et al., 1994); asymmetric cryo-reconstruction of tail machine complex (modified from Lander et al 2009) (Lander et al., 2009); asymmetric cryo-reconstruction of full virion (modified from Lander et al 2006, Science) (Lander et al., 2006).

Figure 2. P22 has an HK97-like body and a telokin-like addition

P22 coat protein in both the immature state “procapsid” (A) and mature state “expanded head” (B) is compared to HK97 in comparable states, “Prohead II” (C), or “Head II” (D). The common structural elements are colored correspondingly; N-arm (red), E-loop (yellow) P-domain (green), A-domain (cyan). P22 has a unique telokin domain colored in magenta. The P22 telokin domain (E) is compared to the telokin domain from myosin light chain kinease “MLCK” (PDB entry 1FHG)(Holden et al., 1992)(F).

Figure 3. Location of tryptophans in P22 coat protein

One subunit of P22 coat protein (procapsid state) with the 6 tryptophan residues shown as spheres and color-coded based on location (B). Same view of the coat protein as in panel A, but including phenylalanines and tyrosines as magenta-colored ball and stick atoms. The location of all of the tryptophans in one asymmetric unit (6 hexon subunits and one penton subunit) in the procapsid state from a top down view. Tryptophans are color-coded according to A. (D) Same as C, but a side view.

Figure 4. Location of conditional lethal variants in P22

Shown in panel A is the primary sequence of P22 with the “backbone” color-coded by domain as in Figure 2 highlighting the 18 tsf mutations (black), 3 cs mutants (purple) and 3 global su mutations (blue). Panel B shows a topology diagram of P22 coat protein obtained through PDBsum (Laskowski et al., 1997) with secondary structural elements represented as cylinders (helices) and arrows (sheets) color coded according to domain. The starting and ending residues in each element are indicated as well as the N and C termini. The orange box designates four β-strands that comprise the β-hinge. C) Ribbon diagram of one subunit with the 17 positions of tsf mutations shown as spheres and color coded according to domain. D) Ribbon diagram of one subunit showing the β-hinge (orange) and the WT residues at the positions of the 3 global su substitutions as cyan spheres.

Figure 5. Kinetic folding model for P22 coat protein

A proposed model for the slow folding reaction is shown. Secondary structure that is not fully formed is represented as dashed tubes for helices and dashed ribbons for sheets. Fully formed secondary structure is represented as solid ribbons and tubes. The unfolded protein rapidly folds to intermediate 1, which has some secondary structure formed and some clustering of tryptophans. During the transition from intermediate 1 to intermediate 2 the tryptophans get fully buried. The intermediate 2 to native state has the formation of remaining secondary structure and complete folding to the native state.

Figure 6. Cs mutants and the electrostatic residues in coat protein that likely dictate scaffolding protein binding

A) Stereo image of one subunit per neighboring hexamer at a true three-fold axis of symmetry shown as a ribbon diagram viewed from inside the procapsid. The N-arm is colored in yellow, with the acidic residues in the N-arm represented as red spheres. The 3 cs mutations are also shown as spheres (T10I in blue, R101C in black, N414S in green). B) Same as panel A, except for the mature state (expanded head).