Molecular Genetics of Bacteriophage P22 Scaffolding Protein's Functional Domains

Weigele, Peter; Sampson, Laura; Winn-Stapley, Danella A.; Casjens, Sherwood

doi:10.1016/j.jmb.2005.03.004

Cited by 67 publications

(90 citation statements)

References 62 publications

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“…1 E and F), which interact with the positively charged C-terminal domain of the scaffolding protein (28). These electrostatic interactions are consistent with previous biochemical studies (31,32). Note that the interactions between the scaffolding protein and coat protein have never been visualized in such fine detail and modeled in any authentic bacteriophage procapsid, although previous attempts at lower resolutions were made in herpes virus capsid (33) and P22 (17).…”

Section: Resultssupporting

confidence: 85%

“…The lack of scaffolding proteins results in the failure to incorporate the portal and can lead to incomplete particles (29,32,40,45). Based on our density maps, we propose that the formation of a unique portal complex with the requisite scaffolding and coat proteins is likely the key for initiating proper procapsid assembly (Fig.…”

Section: Molecular Mechanism For Capsid Assembly Scaffolding Proteinmentioning

confidence: 89%

See 1 more Smart Citation

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Chen

Baker

Hryc

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

202

319

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Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at 3.8-and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry. The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating the capsid assembly at the portal vertex and propagating its growth on a T ¼ 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation into infectious virions.sDNA viruses infecting both prokaryotes and eukaryotes share a common assembly pathway proceeding from a precursor (procapsid) to an infectious virion (1-4). In addition to the coat proteins, the procapsid requires scaffolding proteins, absent from the virion, for proper assembly, and a portal for DNA packaging and subsequent DNA ejection. However, despite a half-century of research on icosahedral viruses, it remains unclear how initially identical subunits adopt both hexameric and pentameric conformations in the virus and select the correct locations needed to form closed shells of the proper size (5). Packaging of DNA through the portal is accompanied by the exit of scaffolding proteins from the procapsid and conformational changes in the coat proteins as the capsid matures (2, 6).Understanding the molecular mechanisms of dsDNA virus assembly and maturation requires knowledge of the interactions among the coat, scaffolding, and portal proteins, all of which are essential for these processes. X-ray crystallography (7-9) and electron cryomicroscopy (cryo-EM) (10-12) have yielded nearatomic to atomic resolution models of several dsDNA icosahedral viruses and provided a structural framework of interactions among their coat proteins. However, the structural details of procapsid portal incorporation, scaffolding protein bind...

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Section: Resultssupporting

confidence: 85%

Section: Molecular Mechanism For Capsid Assembly Scaffolding Proteinmentioning

confidence: 89%

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Chen

Baker

Hryc

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

202

319

View full text Add to dashboard Cite

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“…Interactions between the A-domain of coat protein and scaffolding protein will be the subject of a different study. Since we know that the critical residues in scaffolding protein are basic (R293 and K296), we concentrated our efforts on acidic residues in coat protein (17)(18)(19)26). Salmonella strains transformed with plasmids that express coat protein with single substitutions in the N-arm (E5A, D14A, E15A, and E18A) or the P-loop (K377A and D385A) were infected with P22 phage carrying an amber mutation in gene 5 (codes for coat protein) and plated at different temperatures ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Coat protein binding occurs through scaffolding protein's C-terminal domain, at a helix-turn-helix motif (HTH) where about 30% of the residues are positively charged, indicating that the scaffolding protein-coat protein interaction is mostly electrostatic (17)(18)(19)44). It exists in a monomer-dimertetramer equilibrium in solution, from which the dimer is the most active oligomer during PC nucleation (15).…”

mentioning

confidence: 99%

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

Cortines

Motwani

Vyas

et al. 2014

J Virol

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Icosahedral virus assembly requires a series of concerted and highly specific protein-protein interactions to produce a proper capsid. In bacteriophage P22, only coat protein (gp5) and scaffolding protein (gp8) are needed to assemble a procapsid-like particle, both in vivo and in vitro. In scaffolding protein's coat binding domain, residue R293 is required for procapsid assembly, while residue K296 is important but not essential. Here, we investigate the interaction of scaffolding protein with acidic residues in the N-arm of coat protein, since this interaction has been shown to be electrostatic. Through site-directed mutagenesis of genes 5 and 8, we show that changing coat protein N-arm residue 14 from aspartic acid to alanine causes a lethal phenotype. Coat protein residue D14 is shown by cross-linking to interact with scaffolding protein residue R293 and, thus, is intimately involved in proper procapsid assembly. To a lesser extent, coat protein N-arm residue E18 is also implicated in the interaction with scaffolding protein and is involved in capsid size determination, since a cysteine mutation at this site generated petite capsids. The final acidic residue in the N-arm that was tested, E15, is shown to only weakly interact with scaffolding protein's coat binding domain. This work supports growing evidence that surface charge density may be the driving force of virus capsid protein interactions. IMPORTANCEBacteriophage P22 infects Salmonella enterica serovar Typhimurium and is a model for icosahedral viral capsid assembly. In this system, coat protein interacts with an internal scaffolding protein, triggering the assembly of an intermediate called a procapsid. Previously, we determined that there is a single amino acid in scaffolding protein required for P22 procapsid assembly, although others modulate affinity. Here, we identify partners in coat protein. We show experimentally that relatively weak interactions between coat and scaffolding proteins are capable of driving correctly shaped and sized procapsids and that the lack of these proper protein-protein interfaces leads to aberrant structures. The present work represents an important contribution supporting the hypothesis that virus capsid assembly is governed by seemingly simple interactions. The highly specific nature of the subunit interfaces suggests that these could be good targets for antivirals.

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“…As seen with the P22, P2, 29, and herpes simplex virus (HSV) internal scaffolding proteins (18)(19)(20)(21)(22), the C terminus of the X174 B protein is most strongly associated with the coat protein (23)(24)(25). In the X174 procapsid crystal structure, six aromatic amino acid residues mediate approximately 85% (22/26) of these interactions.…”

mentioning

confidence: 99%

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

Gordon

Fane

2013

J Virol

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C-terminal, aromatic amino acids in the X174 internal scaffolding protein B mediate conformational switches in the viral coat protein. These switches direct the coat protein through early assembly. In addition to the aromatic amino acids, two acidic residues, D111 and E113, form salt bridges with basic, coat protein side chains. Although salt bridge formation did not appear to be critical for assembly, the substitution of an aromatic amino acid for D111 produced a lethal phenotype. This side chain is uniquely oriented toward the center of the coat-scaffolding binding pocket, which is heavily dominated by aromatic ring-ring interactions. Thus, the D111Y substitution may restructure pocket contacts. Previously characterized B ؊ mutants blocked assembly before procapsid formation. However, the D111Y mutant produced an assembled particle, which contained the structural and external scaffolding proteins but lacked protein B and DNA. A suppressor within the external scaffolding protein, which mediates the later stages of particle morphogenesis, restored viability. The unique formation of a postprocapsid particle and the novel suppressor may be indicative of a novel B protein function. However, genetic data suggest that the particle represents the delayed manifestation of an early assembly error. This seemingly late-acting defect was rescued by previously characterized suppressors of early, preprocapsid, B ؊ assembly mutations, which act on the level of coat protein flexibility. Likewise, the newly isolated suppressor in the external scaffolding protein also exhibited a global suppressing phenotype. Thus, the off-pathway product isolated from infected cells may not accurately reflect the temporal nature of the initial defect.

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Molecular Genetics of Bacteriophage P22 Scaffolding Protein's Functional Domains

Cited by 67 publications

References 62 publications

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

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

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

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