Determinants of bacteriophage P22 polyhead formation: the role of coat protein flexibility in conformational switching.
We have investigated determinants of polyhead formation in bacteriophage P22 in order to understand the molecular mechanism by which coat protein assembly goes astray. Polyhead assembly is caused by amino acid substitutions in coat protein at position 170, which is located in the β-hinge. In vivo scaffolding protein does not correct polyhead assembly by F170A or F170K coat proteins, but does for F170L. All F170 variants bind scaffolding protein more weakly than wild-type as observed by affinity chromatography with scaffolding protein-agarose and scaffolding protein shell re-entry experiments. Electron cryo-microscopy and three-dimensional image reconstructions of F170A and F170K empty procapsid shells showed that there is a decreased flexibility of the coat subunits relative to wild-type. This was confirmed by limited proteolysis and protein sequencing, which showed increased protection of the A-domain. Our data support the conclusion that the decrease in flexibility of the A-domain leads to crowding of the subunits at the centre of the pentons, thereby favouring the hexon configuration during assembly. Thus, correct coat protein interactions with scaffolding protein and maintenance of sufficient coat protein flexibility are crucial for proper P22 assembly. The coat protein β-hinge region is the major determinant for both features.
Suhanovsky
Margaret M
MM
Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA.
Parent
Kristin N
KN
Dunn
Sarah E
SE
Baker
Timothy S
TS
Teschke
Carolyn M
CM
D019943:Amino Acid Substitution; D017100:Bacteriophage P22 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D021621:Imaging, Three-Dimensional; D046529:Microscopy, Electron, Transmission; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D019065:Virus Assembly
D036022:Capsid Proteins