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

Padilla-Meier, G. Pauline; Gilcrease, Eddie B.; Weigele, Peter; Cortines, Juliana R.; Siegel, M; Leavitt, Justin C.; Teschke, Carolyn M.; Casjens, Sherwood

doi:10.1074/jbc.m112.393132

Cited by 26 publications

(34 citation statements)

References 80 publications

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“…The essential charged residues are located in the C-terminal helix. However, mutations within the N-terminal helix and the intervening ␤-turn can alter assembly kinetics and eliminate viability without grossly disrupting domain structure or protein binding (26). These observations suggest that some P22 residues perform a subsidiary role, optimizing the architecture of the coat-scaffolding interface.…”

Section: Discussionmentioning

confidence: 91%

“…Thus, in X174, charged residues do not strongly govern coatscaffolding interactions as observed in bacteriophage P22 (4,6,(26)(27)(28). The coat protein-binding domain of the P22 scaffolding protein has a helix-turn-helix (HTH) motif (21).…”

Section: Discussionmentioning

confidence: 99%

“…Electrostatic interactions govern morphogenesis in other viral systems (3) and have been thoroughly studied in bacteriophage P22 (4,6,(26)(27)(28). In the P22 scaffolding protein, two charged residues are central to coat protein binding, whereas other amino acids modulate activity by promoting the proper architecture of the coat-scaffolding protein interface (4,26). Alteration of an amino acid that mediates a protein-protein interaction, either via a salt bridge or via an aromatic ring, is typically regarded as a loss-of-function, or loss-ofcontact, mutation.…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…4C), indicating that these structures are made of ORF62, the major capsid protein. Such open spiral structures were observed for the scaffolding protein-less mutants of phages lambda, P22, and phi2948495051. Binding of the anti-ORF67 antibody to the ΔORF65 mutant was not detected (data not shown).…”

Section: Resultsmentioning

confidence: 88%

Genes essential for the morphogenesis of the Shiga toxin 2-transducing phage from Escherichia coli O157:H7

Mondal

Islam

Sawaguchi

et al. 2016

Sci Rep

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Shiga toxin 2 (Stx2), one of the most important virulence factors of enterohaemorrhagic Escherichia coli (EHEC), is encoded by phages. These phages (Stx2 phages) are often called lambda-like. However, most Stx2 phages are short-tailed, thus belonging to the family Podoviridae, and the functions of many genes, especially those in the late region, are unknown. In this study, we performed a systematic genetic and morphological analysis of genes with unknown functions in Sp5, the Stx2 phage from EHEC O157:H7 strain Sakai. We identified nine essential genes, which, together with the terminase genes, determine Sp5 morphogenesis. Four of these genes most likely encoded portal, major capsid, scaffolding and tail fiber proteins. Although exact roles/functions of the other five genes are unknown, one was involved in head formation and four were required for tail formation. One of the four tail genes encoded an unusually large protein of 2,793 amino-acid residues. Two genes that are likely required to maintain the lysogenic state were also identified. Because the late regions of Stx2 phages from various origins are highly conserved, the present study provides an important basis for better understanding the biology of this unique and medically important group of bacteriophages.

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“…The microvirus internal scaffolding protein shares many properties and functions with the scaffolding proteins found in other viruses (7,9,10,14,15,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). However, it does not physically construct the procapsid or control morphogenetic fidelity.…”

Section: Discussionmentioning

confidence: 99%

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro

Cherwa

Tyson

Bedwell

et al. 2017

J Virol

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During X174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro. In previous studies, X174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. IMPORTANCE Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo. We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During X174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity.KEYWORDS bacteriophage X174, Microviridae, microvirus, scaffolding protein, virus assembly D ue to their rapid replication cycle, detailed structural information, and well developed genetic and biochemical assays, the microviruses have become an ideal model system for experimental evolution (1-4). The morphogenetic pathway can be characterized under restrictive conditions and adaptive mutations can be mapped onto X-ray structures, providing insights into evolutionary mechanisms and protein structure-function relationships (2-8).

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

Cited by 26 publications

References 80 publications

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

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

Genes essential for the morphogenesis of the Shiga toxin 2-transducing phage from Escherichia coli O157:H7

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro

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