Polyhead formation in phage P22 pinpoints a region in coat protein required for conformational switching

Parent, Kristin N.; Suhanovsky, Margaret M.; Teschke, Carolyn M.

doi:10.1111/j.1365-2958.2007.05868.x

Cited by 28 publications

(47 citation statements)

References 57 publications

(89 reference statements)

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“…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). This variant forms tubes in the absence of scaffolding protein both in vivo and in vitro (29).…”

Section: Resultsmentioning

confidence: 99%

“…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).…”

Section: Discussionmentioning

confidence: 99%

“…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). Substitution at F170 leads to increased rigidity of the A domain, which decreases penton formation and results in tubes of coat protein arranged in arrays of hexons with altered orientation at the 3-fold symmetry axis (30,31). This region of coat protein is also thought to be important for interaction with scaffolding protein (7,29,30).…”

Section: Discussionmentioning

confidence: 99%

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

D'Lima

Teschke

2015

J Virol

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Bacteriophage P22, a double-stranded DNA (dsDNA) virus, has a nonconserved 124-amino-acid accessory domain inserted into its coat protein, which has the canonical HK97 protein fold. This I domain is involved in virus capsid size determination and stability, as well as protein folding. The nuclear magnetic resonance (NMR) solution structure of the I domain revealed the presence of a D-loop, which was hypothesized to make important intersubunit contacts between coat proteins in adjacent capsomers. Here we show that amino acid substitutions of residues near the tip of the D-loop result in aberrant assembly products, including tubes and broken particles, highlighting the significance of the D-loops in proper procapsid assembly. Using disulfide cross-linking, we showed that the tips of the D-loops are positioned directly across from each other both in the procapsid and the mature virion, suggesting their importance in both states. Our results indicate that D-loop interactions act as "molecular staples" at the icosahedral 2-fold symmetry axis and significantly contribute to stabilizing the P22 capsid for DNA packaging. Double-stranded DNA (dsDNA) viruses, such as the tailed phages and herpesviruses, have coat proteins that lack sequence homology but possess a common HK97 fold (1, 2). Herpesviruses infect vertebrate hosts and in humans are responsible for a wide spectrum of diseases causing conditions ranging from cold sores and genital sores to chicken pox and shingles. Thus, understanding the assembly of herpesviruses is crucial for identification of novel drug targets. Bacteriophage P22 is a dsDNA virus with a scaffolding protein-mediated assembly pathway, which is similar to that of herpesvirus. Therefore, P22 provides a good simple model system for studying the capsid assembly of dsDNA viruses (3, 4).In bacteriophage P22, capsid assembly involves interaction of 415 coat protein monomers with ϳ100 molecules of scaffolding protein (5-9). This results in the formation of an intermediate structure called the procapsid (10), into which ejection proteins (11, 12) as well as a portal complex are coassembled (13). DNA gets packaged through the portal complex (5), scaffolding protein exits, and the capsid expands in volume (14). The addition of plug, tail needle, and tailspike proteins results in a mature infectious virion (15).Some coat proteins having the HK97 fold are embellished with extra domains (Fig. 1) (16). P22 coat protein is one of these, as it has a nonconserved accessory domain inserted between the A and P domains (10,17,18). This domain, referred to as the insertion domain (I domain) (19), plays an important role in the folding of coat protein by acting as an intramolecular chaperone (20). The I domain is also involved in determination of capsid size (21) and is hypothesized to stabilize procapsids by forming intersubunit interactions between adjacent capsomers (10,19,20). A recent nuclear magnetic resonance (NMR) solution structure of the isolated I domain revealed a large flexible loop, referred to...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

D'Lima

Teschke

2015

J Virol

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“…In addition, mutations that reduce mobility in parts of the protein essential for activity could result in cold sensitivity. These mutants are rescued by the increased thermal energy at higher temperatures (17,(24)(25)(26). There are also situations in which kinetic traps result in misfolding or misassembly at lower temperatures, but get resolved at higher temperatures.…”

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confidence: 94%

“…Using a combination of both ts and cs mutants causing defects at different stages of the assembly pathway, the order of the various steps occurring along this pathway has been determined (14). Thus, these conditional mutants have led to a greater understanding of phage genetics, protein folding, and macromolecular assembly (15)(16)(17). Similarly, cs and ts mutants of different genes essential for cell division, in combination with temperature-shift experiments, have been used to understand the cell cycle phases in which each of these genes act (18).…”

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Rational elicitation of cold-sensitive phenotypes

Baliga

Majhi

Mondal

et al. 2016

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

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Cold-sensitive phenotypes have helped us understand macromolecular assembly and biological phenomena, yet few attempts have been made to understand the basis of cold sensitivity or to elicit it by design. We report a method for rational design of cold-sensitive phenotypes. The method involves generation of partial loss-of-function mutants, at either buried or functional sites, coupled with selective overexpression strategies. The only essential input is amino acid sequence, although available structural information can be used as well. The method has been used to elicit cold-sensitive mutants of a variety of proteins, both monomeric and dimeric, and in multiple organisms, namely Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster. This simple, yet effective technique of inducing cold sensitivity eliminates the need for complex mutations and provides a plausible molecular mechanism for eliciting cold-sensitive phenotypes.conditional mutants | rational design | cold sensitivity | heat-induced expression | transfer between organisms C onditional mutants are powerful tools for studying gene function in vivo. A conditional mutant retains the function of a gene under one set of conditions, called permissive, and lacks that function under a different set of conditions, called restrictive, whereas the wild type (WT) phenotype is similar across both conditions. Cold-sensitive (cs) mutants behave like loss-of-function mutants at temperatures lower than a cutoff temperature, but have WT-like phenotypes at higher temperatures. In contrast, temperature-sensitive (ts) mutants show heat sensitivity and behave like the WT below the restrictive temperature. Both cs and ts mutants can provide important information about protein structure, function, and assembly. cs mutants are rarer than ts mutants, and the molecular basis for generation of cs phenotypes is currently unclear.cs mutants have been used to analyze various biological phenomena, most commonly the cell cycle (1-3), ribosome assembly (4-8), and protein export (9-11), as well as to understand macromolecular structure-function relationships (12, 13). Various ts and cs variants also have helped us understand P22 phage coat protein assembly. Using a combination of both ts and cs mutants causing defects at different stages of the assembly pathway, the order of the various steps occurring along this pathway has been determined (14). Thus, these conditional mutants have led to a greater understanding of phage genetics, protein folding, and macromolecular assembly (15)(16)(17). Similarly, cs and ts mutants of different genes essential for cell division, in combination with temperature-shift experiments, have been used to understand the cell cycle phases in which each of these genes act (18).Various changes at the amino acid level or gene regulation level can cause cold sensitivity. Some cs mutants have been suggested to result from altered feedback inhibition of certain metabolic pathways at lower temperatures (19,20). Cold sensitivity also has been attribut...

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