Functional domains of the bacteriophage P2 scaffolding protein: identification of residues involved in assembly and protease activity.
Bacteriophage P2 encodes a scaffolding protein, gpO, which is required for correct assembly of P2 procapsids from the gpN major capsid protein. 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. 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 protease activity of gpO resides in the N-terminal half of the protein. We show that gpO is a classical serine protease, with a catalytic triad comprised of Asp 19, His 48 and Ser 107. The C-terminal 90 amino acids of gpO are required and sufficient for capsid assembly. This fragment contains a predicted alpha-helical segment between residues 197 and 257 and exists as a multimer in solution, suggesting that oligomerization is required for scaffolding activity. Correct assembly requires the C-terminal cysteine residue, which is most likely involved in transient gpN interactions. 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 alpha-helical domain of gpO and transient interactions between Cys 284 and gpN lead to capsid assembly.
Chang
Jenny R
JR
Department of Microbiology, University of Alabama at Birmingham, BBRB 311, Birmingham, AL 35294, USA.
Spilman
Michael S
MS
Rodenburg
Cynthia M
CM
Dokland
Terje
T
D017901:Bacteriophage P2 / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D002213:Capsid; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D004279:DNA, Viral / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*
D036022:Capsid Proteins; D004279:DNA, Viral; D012330:RNA, Double-Stranded; D015678:Viral Structural Proteins; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases