![]() Selected gpO truncation constructs that were made and the segments of α-helical structure included in each are shown in the boxes below. The predicted α-helical structures are shown as squiggly lines. The sequence of gpO from 1–284 is represented by the rectangular box at the top. Such an α-helical nature is similar to the scaffolding proteins of other viruses such as P22 and φ29 ( Morais et al., 2003 Sun et al., 2000).ĭesign of gpO truncation constructs. This fragment includes a long predicted αhelical sequence between residues 197 and 257 ( Fig. Additionally, a C-terminal fragment that includes only residues 195–284 is able to promote the formation of correctly formed P2 procapsids ( Chang et al., 2008). The truncated protein is capable of catalyzing the formation of P2 procapsid-like particles upon co-expression with gpN. coli however, truncation of the first 25 resides from the N-terminus of the protein abolishes the proteolytic activity ( Wang et al., 2006), suggesting that one of the active site residues for the protease activity resides within these 25 residues. The volume of the P4 capsid only permits encapsidation of the smaller P4 genome.įull-length gpO undergoes autoproteolytic degradation upon expression in E. The size of the P4 capsid is determined by the P4-encoded Sid protein, which forms an external scaffold around the P4 procapsids ( Barrett et al., 1976 Marvik et al., 1995). In the presence of the genetically unrelated bacteriophage P4, 235 copies of gpN-derived protein are assembled into a smaller, 45 nm capsid with T=4 symmetry ( Dokland et al., 1992 Lindqvist et al., 1993). Capsid maturation also involves processing of gpN into N* through the removal of the N-terminal 31 residues ( Rishovd and Lindqvist, 1992). Following assembly, gpO is cleaved autoproteolytically between residues 141 and 142 into a 15.5 kDa N-terminal fragment, O*, that remains in the mature capsid ( Chang et al., 2008 Lengyel et al., 1973 Rishovd and Lindqvist, 1992). Formation of viable procapsids in addition requires the assembly of 12 copies of the portal protein gpQ into a ring-like connector at a unique vertex of the capsid, through which the DNA is packaged and to which the tail is subsequently attached ( Doan and Dokland, 2007 Rishovd et al., 1994). Assembly of the 55 nm P2 procapsid is dependent on the 31.7 kDa scaffolding protein gpO, which also acts as a protease during capsid maturation ( Chang et al., 2008 Lengyel et al., 1973 Wang et al., 2006). The protease gene usually precedes the scaffolding protein gene in the genome ( Hendrix, 2003).īacteriophage P2 forms a 60 nm diameter, T=7 icosahedral capsid from 415 copies of a 36.7 kDa capsid protein (N*) derived from gene product N (gpN) ( Dokland et al., 1992 Lindqvist et al., 1993). Many bacteriophages also encode a protease that is involved in processing the structural proteins during capsid maturation. The scaffolding protein gene typically resides in the same operon as the major capsid protein gene, immediately preceding it in the phage genome. Some scaffolding proteins act as direct catalysts of the assembly process, while others serve more to prevent incorrect interactions, or affect the assembly kinetics ( Dokland, 1999 Fane and Prevelige, 2003). These proteins act as chaperones for the assembly process, but are degraded or removed from the head during capsid maturation and DNA packaging. Most of the known double-stranded (ds) DNA bacteriophages use scaffolding proteins for the assembly of their capsids. Our results suggest a model for gpO scaffolding action in which the N-terminal half of gpO binds strongly to gpN, while oligomerization of the C-terminal α-helical domain of gpO and transient interactions between Cys 284 and gpN lead to capsid assembly. Correct assembly requires the C-terminal cysteine residue, which is most likely involved in transient gpN interactions. This fragment contains a long α-helical segment between residues 197 and 257 and exists as a multimer in solution, suggesting that oligomerization is required for scaffolding activity. The C-terminal 90 amino acids of gpO are required and sufficient for capsid assembly. We show that gpO is a classical serine protease, with a catalytic triad comprised of Asp 19, His 48 and Ser 107. The protease activity of gpO resides in the N-terminal half of the protein. In addition, gpO is presumed to act as the maturation protease for gpN, which is N-terminally processed to N*, accompanied by DNA packaging and capsid expansion. The 284 residue gpO protein also acts as a protease, cleaving itself into an N-terminal fragment, O*, that remains in the capsid following maturation. Bacteriophage P2 encodes a scaffolding protein, gpO, which is required for correct assembly of P2 procapsids from the gpN major capsid protein. ![]()
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