Determinants of bacteriophage P22 polyhead formation: the role of coat protein flexibility in conformational switching

Abstract

SummaryWe 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 b-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 cryomicroscopy 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 b-hinge region is the major determinant for both features.

Effects of an Early Conformational Switch Defect during ϕX174 Morphogenesis Are Belatedly Manifested Late in the Assembly Pathway

“…While the absence of coat protein binding can be easily distinguished from other defects (4)(5)(6), the fluid nature of assembly can obscure the differences between defects in the later stages. An altered nucleation complex could result in the formation of an aberrantly shaped assembled particle (4,7).…”

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

“…This variant forms tubes in the absence of scaffolding protein both in vivo and in vitro (29). However, in the presence of scaffolding protein, tube formation of F170L coat protein can be decreased or eliminated (29). Since D246A coat protein formed tubes in infected cells in vivo (with scaffolding protein present), we asked if this coat protein variant was also able to generate tubes in vitro, as was observed with the F170L coat protein variant.…”

“…In vivo, the absence of scaffolding protein leads to assembly of coat protein into aberrant structures, including petite T ϭ 4 capsids and spirals (25)(26)(27)(28). 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).…”

“…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). However, in the presence of scaffolding protein, tube formation of F170L coat protein can be decreased or eliminated (29).…”

“…In contrast, D246A coat protein lost the ability to assemble into proper procapsids, but rather, it assembles into tubes in vivo. Tube formation of P22 coat protein has been seen previously only when substitutions of phenylalanine at position 170 were made (29,30). Position F170 is located in the ␤-hinge of coat protein's A domain, a region shown to be important for conformational switching during maturation (29,37).…”