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Functional domains of bacteriophage P22 scaffolding protein
M H Parker1, S Casjens, P E Prevelige
1Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA.
Abstract:
Assembly of the bacteriophage P22 requires a 303 amino acid residue scaffolding protein. Two scaffolding protein deletion mutants, consisting of residues 141 to 303 and 141 to 292, have been described. We report here that the 141-303 fragment, but not the 141-292 fragment, promoted procapsid assembly in vitro, bound to preformed shells of coat protein, and bound to a coat protein affinity column. These findings suggest that the carboxyl-terminal half of the scaffolding protein is sufficient for promoting assembly, and that the 11 amino acid residues at the extreme carboxyl terminus are required for binding to the coat protein. Analysis of the products of in vitro assembly reactions suggests that the maximum amount of scaffolding protein that can pack into a procapsid is dictated by the internal volume of the procapsid rather than by a finite number of binding sites. However, when the amount of scaffolding protein was reduced to limiting values, both the wild-type protein and the 141-303 fragment assembled procapsids with the same number, rather than the same mass, of scaffolding protein molecules. When the 141-292 fragment was added to a mixture of coat and scaffolding proteins, the initial phase of procapsid assembly was inhibited, but the final yield and composition of the procapsids were not affected. Assembly by a covalent dimeric mutant scaffolding protein (R74C/L177I) was not inhibited by the 141-292 fragment, which suggests that the inhibition is due to the formation of inactive heterodimers between the 141-292 fragment and the monomeric scaffolding protein. The 141-303 fragment, which has less tendency to self-associate than the wild-type protein, formed aberrant species as well as normal procapsid-like particles when the rate of assembly was high, suggesting that scaffolding protein dimerization may play a role in ensuring fidelity of assembly. Alternatively, residues 1 to 140 may play a direct structural role in preventing inappropriate scaffolding/coat protein interactions.
Insights
The carboxyl-terminal half of bacteriophage P22 scaffolding protein drives procapsid assembly and coat protein binding. The final 11 residues are crucial for this interaction, influencing assembly fidelity.
Area of Science:
- Structural biology
- Virology
- Molecular assembly
Background:
- Bacteriophage P22 procapsid assembly relies on a scaffolding protein.
- Previous studies identified two deletion mutants of this protein.
Purpose of the Study:
- To investigate the functional domains of the P22 scaffolding protein.
- To determine the role of specific residues in procapsid assembly and coat protein interaction.
Main Methods:
- In vitro assembly assays using wild-type and mutant scaffolding proteins.
- Coat protein affinity chromatography.
- Analysis of procapsid composition and assembly products.
Main Results:
- The 141-303 fragment promoted assembly and bound coat protein, unlike the 141-292 fragment.
- The extreme C-terminal 11 residues are essential for coat protein binding.
- Procapsid internal volume, not binding sites, dictates maximum scaffolding protein incorporation.
- The 141-292 fragment inhibited assembly by forming inactive heterodimers with wild-type scaffolding protein.
Conclusions:
- The carboxyl-terminal half of the scaffolding protein is sufficient for promoting assembly.
- Specific C-terminal residues are critical for coat protein interaction.
- Scaffolding protein dimerization may be important for assembly fidelity.