Bacteriophage P22 capsid size determination: Roles for the coat protein telokin-like domain and the scaffolding protein amino-terminus

Abstract

Assembly of icosahedral capsids of proper size and symmetry is not understood. Residue F170 in bacteriophage P22 coat protein is critical for conformational switching during assembly. Substitutions at this site cause assembly of tubes of hexamerically arranged coat protein. Intragenic suppressors of the ts phenotype of F170A and F170K coat protein mutants were isolated. Suppressors were repeatedly found in the coat protein telokin-like domain at position 285, which caused coat protein to assemble into petite procapsids and capsids. Petite capsid assembly strongly correlated to the side chain volume of the substituted amino acid. We hypothesize that larger side chains at position 285 torque the telokin-like domain, changing flexibility of the subunit and intercapsomer contacts. Thus, a single amino acid substitution in coat protein is sufficient to change capsid size. In addition, the products of assembly of the variant coat proteins were affected by the size of the internal scaffolding protein.

Highly Specific Salt Bridges Govern Bacteriophage P22 Icosahedral Capsid Assembly: Identification of the Site in Coat Protein Responsible for Interaction with Scaffolding Protein

“…In solution, the N-terminal and the C-terminal domains seem to be in close proximity, which is not true for the scaffolding protein population present inside procapsids (17,25). Interestingly, the N-terminal domain of scaffolding protein, to a lesser extent, also affects the interaction of scaffolding protein and coat protein, possibly acting as a modulator to reduce the affinity between these two proteins (25)(26)(27). Recently, we determined the specific sites of interaction of scaffolding protein with coat protein.…”

Unraveling the Role of the C-terminal Helix Turn Helix of the Coat-binding Domain of Bacteriophage P22 Scaffolding Protein

“…The scaffolding-like ␦ domain of the HK97 major capsid protein and the internal scaffolding protein of ⌽X174 both appear to function by directing a switch of the major capsid protein domains during assembly (19 -21). It is not known if the free scaffolding proteins of the large viruses may use a similar mechanism; however, Suhanovsky and Teschke (22) have argued that phage P22 scaffolding protein also affects conformational switching in the major capsid protein during assembly.…”

“…Although electrostatic interactions, via the two critical amino acids in the scaffolding protein HTH, are a primary factor responsible for this interaction, the exact shape of the HTH might also be crucial. The scaffolding protein is folded so that the N terminus, which also plays a role in assembly (22), is positioned in the immediate vicinity of the C terminus, and it is not a rigid molecule because it requires flexibility of some domains to function properly (51).…”

A Molecular Staple: D-Loops in the I Domain of Bacteriophage P22 Coat Protein Make Important Intercapsomer Contacts Required for Procapsid Assembly

“…In previous work, we showed the coat protein variant F170L, like D246A coat protein, formed tubes (29). The position F170 is located in the ␤-hinge region of the coat protein core and is a fourstranded ␤-sheet important for conformational switching during assembly and capsid maturation (21,30,31). This variant forms tubes in the absence of scaffolding protein both in vivo and in vitro (29).…”

“…This domain, referred to as the insertion domain (I domain) (19), plays an important role in the folding of coat protein by acting as an intramolecular chaperone (20). The I domain is also involved in determination of capsid size (21) and is hypothesized to stabilize procapsids by forming intersubunit interactions between adjacent capsomers (10,19,20). A recent nuclear magnetic resonance (NMR) solution structure of the isolated I domain revealed a large flexible loop, referred to as the D-loop (18,19,22).…”