Identification of a second site for coat protein binding in bacteriophage P22 scaffolding protein

Abstract

Scaffolding proteins are essential for the assembly of most tailed, double-stranded DNA bacteriophages as well as herpesviruses. These proteins interact specifically with the coat proteins to efficiently assemble procapsids with the correct morphology. A helix-turn-helix (HTH) domain of bacteriophage P22 scaffolding protein is essential for coat binding, but the presence of additional coat protein binding sites has been predicted. An alanine substitution at scaffolding protein residue L245 causes a strong cold-sensitive phenotype. Bothin vivoandin vitroassembly with L245A scaffolding protein yields aberrant and petite particles at non-permissive temperatures. The L245A scaffolding protein is destabilized as determined by thermal melts monitored by circular dichroism. Through crosslinking studies, residue L245 interacts with the coat protein A-domain residue D198, which has been predicted previously to contain a scaffolding protein binding site. L245 also binds R101 in the coat protein P-domain as well as E18 in the N-arm. These results demonstrate the presence of secondary coat binding sites that may function in conjunction with the HTH domain to promote the assembly of procapsids with the correct curvature.ImportanceMany dsDNA viruses, including tailed bacteriophages and Herpesviruses, assemble precursor capsids, or procapsids, using an essential catalytic scaffolding protein. How scaffolding proteins induce proper assembly of their major capsid proteins remains unclear. The scaffolding protein of bacteriophage P22 has a C-terminal helix-turn-helix domain that interacts with the N-arm of the coat protein to activate it for assembly. Here, a second potential coat protein interaction site is identified in scaffolding protein at residue L245. Residue L245 may be involved in stabilizing a small helical domain based the effect of the substitution scaffolding protein stability and a predicted model of the scaffolding protein fold, thereby indicating additional complexity in the interaction between coat and scaffolding proteins.