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Roger W. Hendrix - One of the best experts on this subject based on the ideXlab platform.
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Capsids and Genomes of Jumbo-Sized Bacteriophages Reveal the Evolutionary Reach of the HK97 Fold.
mBio, 2017Co-Authors: Jianfei Hua, Robert L. Duda, Roger W. Hendrix, Welkin H. Pope, Alexis Huet, Carlos A. Lopez, Katerina Toropova, James F. ConwayAbstract:Large icosahedral viruses that infect bacteria represent an extreme of the coevolution of capsids and the genomes they accommodate. One subset of these large viruses is the jumbophages, tailed phages with double-stranded DNA genomes of at least 200,000 bp. We explored the mechanism leading to increased capsid and genome sizes by characterizing structures of several jumbophage capsids and the DNA packaged within them. Capsid structures determined for six jumbophages were consistent with the canonical phage HK97 fold, and three had capsid geometries with novel triangulation numbers (T=25, T=28, and T=52). Packaged DNA (chromosome) sizes were larger than the genome sizes, indicating that all jumbophages use a head-full DNA packaging mechanism. For two phages (PAU and G), the sizes appeared very much larger than their genome length. We used two-dimensional DNA gel electrophoresis to show that these two DNAs migrated abnormally due to base modifications and to allow us to calculate their actual chromosome sizes. Our results support a ratchet model of capsid and genome coevolution whereby mutations lead to increased capsid volume and allow the acquisition of additional genes. Once the added genes and larger capsid are established, mutations that restore the smaller size are disfavored.IMPORTANCE A large family of viruses share the same fold of the capsid protein as bacteriophage HK97, a virus that infects bacteria. Members of this family use different numbers of the capsid protein to build capsids of different sizes. Here, we examined the structures of extremely large capsids and measured their DNA content relative to the sequenced genome lengths, aiming to understand the process that increases size. We concluded that mutational changes leading to larger capsids become locked in by subsequent changes to the genome organization.
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Flexible Connectors between Capsomer Subunits that Regulate Capsid Assembly.
Journal of molecular biology, 2017Co-Authors: Mary L. Hasek, Roger W. Hendrix, Joshua B. Maurer, Robert L. DudaAbstract:Abstract Viruses build icosahedral capsids of specific size and shape by regulating the spatial arrangement of the hexameric and pentameric protein capsomers in the growing shell during assembly. In the T = 7 capsids of Escherichia coli bacteriophage HK97 and other phages, 60 capsomers are hexons, while the rest are pentons that are correctly positioned during assembly. Assembly of the HK97 capsid to the correct size and shape has been shown to depend on specific ionic contacts between capsomers. We now describe additional ionic interactions within capsomers that also regulate assembly. Each is between the long hairpin, the “E-loop,” that extends from one subunit to the adjacent subunit within the same capsomer. Glutamate E153 on the E-loop and arginine R210 on the adjacent subunit's backbone alpha-helix form salt bridges in hexamers and pentamers. Mutations that disrupt these salt bridges were lethal for virus production, because the mutant proteins assembled into tubes or sheets instead of capsids. X-ray structures show that the E153–R210 links are flexible and maintained during maturation despite radical changes in capsomer shape. The E153–R210 links appear to form early in assembly to enable capsomers to make programmed changes in their shape during assembly. The links also prevent flattening of capsomers and premature maturation. Mutant phenotypes and modeling support an assembly model in which flexible E153–R210 links mediate capsomer shape changes that control where pentons are placed to create normal-sized capsids. The E-loop may be conserved in other systems in order to play similar roles in regulating assembly.
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On the catalytic mechanism of bacteriophage HK97 capsid crosslinking.
Virology, 2017Co-Authors: Danju Tso, Robert L. Duda, Joshua B. Maurer, Craig L. Peebles, Roger W. HendrixAbstract:During maturation of the phage HK97 capsid, each of the 415 capsid subunits forms covalent bonds to neighboring subunits, stabilizing the capsid. Crosslinking is catalyzed not by a separate enzyme but by subunits of the assembled capsid in response to conformational rearrangements during maturation. This report investigates the catalytic mechanism. Earlier work established that the crosslinks are isopeptide (amide) bonds between side chains of a lysine on one subunit and an asparagine on another subunit, aided by a catalytic glutamate on a third subunit. The mature capsid structure suggests that the reaction may be facilitated by the arrival of a valine with the lysine to complete a hydrophobic pocket surrounding the glutamate, lysine and asparagine. We show that this valine has an essential role for efficient crosslinking, and that any of six other amino acids can successfully substitute for valine. Evidently none of the remaining 13 amino acids will work.
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Correct Assembly of the Bacteriophage T5 Procapsid Requires Both the Maturation Protease and the Portal Complex
Journal of Molecular Biology, 2016Co-Authors: Alexis Huet, Robert L. Duda, Roger W. Hendrix, Pascale Boulanger, James F. ConwayAbstract:The 90-nm-diameter capsid of coliphage T5 is organized with T=13 icosahedral geometry and encloses a double-stranded DNA genome that measures 121kbp. Its assembly follows a path similar to that of phage HK97 but yielding a larger structure that includes 775 subunits of the major head protein, 12 subunits of the portal protein and 120 subunits of the decoration protein. As for phage HK97, T5 encodes the scaffold function as an N-terminal extension (∆-domain) to the major head protein that is cleaved by the maturation protease after assembly of the initial prohead I form and prior to DNA packaging and capsid expansion. Although the major head protein alone is sufficient to assemble capsid-like particles, the yield is poor and includes many deformed structures. Here we explore the role of both the portal and the protease in capsid assembly by generating constructs that include the major head protein and a combination of protease (wild type or an inactive mutant) and portal proteins and overexpressing them in Escherichia coli. Our results show that the inactive protease mutant acts to trigger assembly of the major head protein, probably through binding to the ∆-domain, while the portal protein regulates assembly into the correct T=13 geometry. A cryo-electron microscopy reconstruction of prohead I including inactivated protease reveals density projecting from the prohead interior surface toward its center that is compatible with the ∆-domain, as well as additional internal density that we assign as the inactivated protease. These results reveal complexity in T5 beyond that of the HK97 system.
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Metastable Intermediates as Stepping Stones on the Maturation Pathways of Viral Capsids
mBio, 2014Co-Authors: Giovanni Cardone, Robert L. Duda, Roger W. Hendrix, James F. Conway, Naiqian Cheng, Lili You, Alasdair C. StevenAbstract:As they mature, many capsids undergo massive conformational changes that transform their stability, reactivity, and capacity for DNA. In some cases, maturation proceeds via one or more intermediate states. These structures represent local min- ima in a rich energy landscape that combines contributions from subunit folding, association of subunits into capsomers, and intercapsomer interactions. We have used scanning calorimetry and cryo-electron microscopy to explore the range of capsid conformations accessible to bacteriophage HK97. To separate conformational effects from those associated with covalent cross- linking (a stabilization mechanism of HK97), a cross-link-incompetent mutant was used. The mature capsid Head I undergoes an endothermic phase transition at 60°C in which it shrinks by 7%, primarily through changes in its hexamer conformation. The transition is reversible, with a half-life of ~3 min; however, >50% of reverted capsids are severely distorted or ruptured. This observation implies that such damage is a potential hazard of large-scale structural changes such as those involved in matura- tion. Assuming that the risk is lower for smaller changes, this suggests a rationalization for the existence of metastable interme- diates: that they serve as stepping stones that preserve capsid integrity as it switches between the radically different conforma- tions of its precursor and mature states. IMPORTANCE Large-scale conformational changes are widespread in virus maturation and infection processes. These changes are accompanied by the release of conformational free energy as the virion (or fusogenic glycoprotein) switches from a precursor state to its mature state. Each state corresponds to a local minimum in an energy landscape. The conformational changes in cap- sid maturation are so radical that the question arises of how maturing capsids avoid being torn apart. Offering proof of princi- ple, severe damage is inflicted when a bacteriophage HK97 capsid reverts from the (nonphysiological) state that it enters when heated past 60°C. We suggest that capsid proteins have been selected in part by the criterion of being able to avoid sustaining collateral damage as they mature. One way of achieving this—as with the HK97 capsid—involves breaking the overall transition down into several smaller steps in which the risk of damage is reduced.
Robert L. Duda - One of the best experts on this subject based on the ideXlab platform.
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Capsids and Portals Influence Each Other's Conformation During Assembly and Maturation.
Journal of molecular biology, 2020Co-Authors: Joshua B. Maurer, Crystal L. Moyer, Robert L. DudaAbstract:Abstract The portal proteins of tailed bacteriophage and Herpesvirus capsids form dodecameric rings that occupy one capsid vertex and are incorporated during the assembly of capsid precursors called procapsids or proheads. Portals are essential and serve as the pore for DNA transit and the site of tail attachment; however, bacteriophage HK97 capsid proteins assemble efficiently without a portal when expressed from plasmids. Following portal co-expression, portals were incorporated into about half of the proheads that were made. In the absence of active capsid maturation protease, uncleaved proheads formed dimers, trimers, and tetramers of proheads during purification, but only if they had portals. These appeared bound to membrane-like fragments by their portals and could be disaggregated by detergents, supporting a role for membranes in their formation and in capsid assembly. The precursors to prohead oligomers were detected in cell extracts. These were able to bind to Octyl-Sepharose and could be released by detergent, while uncleaved proheads without portal or cleaved proheads with portal did not bind. Our results document a discrete change in the HK97 portal’s hydrophobicity induced by cleavage of the procapsid shell in which it is embedded. Additionally, we detected an increase in the rate of expansion induced by the presence of a portal complex in cleaved HK97 proheads. These results suggest that portals and capsids influence each other’s conformation during assembly. The formation of prohead oligomers also provides a rapid and sensitive assay for identification and analysis of portal incorporation mutants.
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The amazing HK97 fold: versatile results of modest differences
Current opinion in virology, 2019Co-Authors: Robert L. Duda, Carolyn M TeschkeAbstract:dsDNA Bacteriophages, some dsDNA archaeal viruses and the Herpesviruses share many features including a common capsid assembly pathway and coat protein fold. The coat proteins of these viruses, which have the HK97 fold, co-assemble with a free or attached scaffolding protein and other capsid proteins into a precursor capsid, known as a procapsid or prohead. The procapsid is a metastable state that increases in stability as a result of morphological changes that occur during the dsDNA packaging reaction. We review evidence from several systems indicating that proper contacts acquired in the assembly of the procapsid are critical to forming the correct morphology in the mature capsid.
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Capsids and Genomes of Jumbo-Sized Bacteriophages Reveal the Evolutionary Reach of the HK97 Fold.
mBio, 2017Co-Authors: Jianfei Hua, Robert L. Duda, Roger W. Hendrix, Welkin H. Pope, Alexis Huet, Carlos A. Lopez, Katerina Toropova, James F. ConwayAbstract:Large icosahedral viruses that infect bacteria represent an extreme of the coevolution of capsids and the genomes they accommodate. One subset of these large viruses is the jumbophages, tailed phages with double-stranded DNA genomes of at least 200,000 bp. We explored the mechanism leading to increased capsid and genome sizes by characterizing structures of several jumbophage capsids and the DNA packaged within them. Capsid structures determined for six jumbophages were consistent with the canonical phage HK97 fold, and three had capsid geometries with novel triangulation numbers (T=25, T=28, and T=52). Packaged DNA (chromosome) sizes were larger than the genome sizes, indicating that all jumbophages use a head-full DNA packaging mechanism. For two phages (PAU and G), the sizes appeared very much larger than their genome length. We used two-dimensional DNA gel electrophoresis to show that these two DNAs migrated abnormally due to base modifications and to allow us to calculate their actual chromosome sizes. Our results support a ratchet model of capsid and genome coevolution whereby mutations lead to increased capsid volume and allow the acquisition of additional genes. Once the added genes and larger capsid are established, mutations that restore the smaller size are disfavored.IMPORTANCE A large family of viruses share the same fold of the capsid protein as bacteriophage HK97, a virus that infects bacteria. Members of this family use different numbers of the capsid protein to build capsids of different sizes. Here, we examined the structures of extremely large capsids and measured their DNA content relative to the sequenced genome lengths, aiming to understand the process that increases size. We concluded that mutational changes leading to larger capsids become locked in by subsequent changes to the genome organization.
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Flexible Connectors between Capsomer Subunits that Regulate Capsid Assembly.
Journal of molecular biology, 2017Co-Authors: Mary L. Hasek, Roger W. Hendrix, Joshua B. Maurer, Robert L. DudaAbstract:Abstract Viruses build icosahedral capsids of specific size and shape by regulating the spatial arrangement of the hexameric and pentameric protein capsomers in the growing shell during assembly. In the T = 7 capsids of Escherichia coli bacteriophage HK97 and other phages, 60 capsomers are hexons, while the rest are pentons that are correctly positioned during assembly. Assembly of the HK97 capsid to the correct size and shape has been shown to depend on specific ionic contacts between capsomers. We now describe additional ionic interactions within capsomers that also regulate assembly. Each is between the long hairpin, the “E-loop,” that extends from one subunit to the adjacent subunit within the same capsomer. Glutamate E153 on the E-loop and arginine R210 on the adjacent subunit's backbone alpha-helix form salt bridges in hexamers and pentamers. Mutations that disrupt these salt bridges were lethal for virus production, because the mutant proteins assembled into tubes or sheets instead of capsids. X-ray structures show that the E153–R210 links are flexible and maintained during maturation despite radical changes in capsomer shape. The E153–R210 links appear to form early in assembly to enable capsomers to make programmed changes in their shape during assembly. The links also prevent flattening of capsomers and premature maturation. Mutant phenotypes and modeling support an assembly model in which flexible E153–R210 links mediate capsomer shape changes that control where pentons are placed to create normal-sized capsids. The E-loop may be conserved in other systems in order to play similar roles in regulating assembly.
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On the catalytic mechanism of bacteriophage HK97 capsid crosslinking.
Virology, 2017Co-Authors: Danju Tso, Robert L. Duda, Joshua B. Maurer, Craig L. Peebles, Roger W. HendrixAbstract:During maturation of the phage HK97 capsid, each of the 415 capsid subunits forms covalent bonds to neighboring subunits, stabilizing the capsid. Crosslinking is catalyzed not by a separate enzyme but by subunits of the assembled capsid in response to conformational rearrangements during maturation. This report investigates the catalytic mechanism. Earlier work established that the crosslinks are isopeptide (amide) bonds between side chains of a lysine on one subunit and an asparagine on another subunit, aided by a catalytic glutamate on a third subunit. The mature capsid structure suggests that the reaction may be facilitated by the arrival of a valine with the lysine to complete a hydrophobic pocket surrounding the glutamate, lysine and asparagine. We show that this valine has an essential role for efficient crosslinking, and that any of six other amino acids can successfully substitute for valine. Evidently none of the remaining 13 amino acids will work.
John E. Johnson - One of the best experts on this subject based on the ideXlab platform.
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Allosteric effects in bacteriophage HK97 procapsids revealed directly from covariance analysis of cryo EM data.
Journal of structural biology, 2018Co-Authors: David Veesler, Peter C. Doerschuk, John E. JohnsonAbstract:Abstract The information content of cryo EM data sets exceeds that of the electron scattering potential (cryo EM) density initially derived for structure determination. Previously we demonstrated the power of data variance analysis for characterizing regions of cryo EM density that displayed functionally important variance anomalies associated with maturation cleavage events in Nudaurelia Omega Capensis Virus and the presence or absence of a maturation protease in bacteriophage HK97 procapsids. Here we extend the analysis in two ways. First, instead of imposing icosahedral symmetry on every particle in the data set during the variance analysis, we only assume that the data set as a whole has icosahedral symmetry. This change removes artifacts of high variance along icosahedral symmetry axes, but retains all of the features previously reported in the HK97 data set. Second we present a covariance analysis that reveals correlations in structural dynamics (variance) between the interior of the HK97 procapsid with the protease and regions of the exterior (not seen in the absence of the protease). The latter analysis corresponds well with hydrogen deuterium exchange studies previously published that reveal the same correlation.
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Maturation in Action: CryoEM Study of a Viral Capsid Caught during Expansion
Structure (London England : 1993), 2012Co-Authors: David Veesler, Clinton S. Potter, Bridget Carragher, Joel Quispe, Nikolaus Grigorieff, John E. JohnsonAbstract:Summary Bacteriophage HK97 maturation involves discrete intermediate particle forms, comparable to transitional states in protein folding, before reaching its mature form. The process starts by formation of a metastable prohead, poised for exothermic expansion triggered by DNA packaging. During maturation, the capsid subunit transitions from a strained to a canonical tertiary conformation and this has been postulated to be the driving mechanism for initiating expansion via switching hexameric capsomer architecture from skewed to 6-fold symmetric. We report the subnanometer electron-cryomicroscopy reconstruction of the HK97 first expansion intermediate before any crosslink formation. This form displays 6-fold symmetric hexamers, but capsid subunit tertiary structures exhibit distortions comparable to the prohead forms. We propose that coat subunit strain release acts in synergy with the first crosslinks to drive forward maturation. Finally, we speculate that the energetic features of this transition may result from increased stability of intermediates during maturation via enhanced inter-subunit interactions.
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Bacteriophage HK97 Capsid Assembly and Maturation
Advances in experimental medicine and biology, 2011Co-Authors: Roger W. Hendrix, John E. JohnsonAbstract:The Escherichia coli phage HK97 has provided a productive experimental system for investigating how virus capsids are assembled from their protein components and how the assembled capsids mature to their final form. Aspects of the process for which the HK97 system has been particularly informative include assembly of the icosahedral capsid shell from the component proteins, structure of the capsid subunits and of the entire capsid as it progresses through its maturation, and the mechanism of the covalent cross-linking that links the subunits together into viral chain mail. The structural dynamics of the maturation as well as the energetics that drives maturation forward are beginning to be understood.
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Transferrin-mediated targeting of bacteriophage HK97 nanoparticles into tumor cells.
Nanomedicine (London England), 2011Co-Authors: Rick K. Huang, Nicole F. Steinmetz, Marianne Manchester, John E. JohnsonAbstract:Aims: Next-generation targeted nanodevices are currently under development for imaging and therapeutic applications. We engineered HK97 viral nanoparticles (VNPs) for tumor cell-specific targeting. Methods: A combination of genetic and chemical engineering methods were developed and applied to generate dual-labeled HK97 cysteine mutant particles displaying transferrin and fluorescent labels. The targeting properties of transferrin-conjugated VNPs were evaluated by in vitro experiments using different cancer cell lines. Results: We found that HK97–tranferrin formulations were indeed targeted to cancer cells in vitro via the transferrin receptor. These studies highlight the utility and facilitate the further development of HK97-based VNPs.
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virus particle maturation insights into elegantly programmed nanomachines
Current Opinion in Structural Biology, 2010Co-Authors: John E. JohnsonAbstract:Similar modes of virus maturation have been observed in dsDNA bacteriophages and the structurally related herpes viruses and some type of maturation occur in most animal viruses. Recently a variety of biophysical studies of maturation intermediates of bacteriophages P22, λ, and HK97 have suggested an energy landscape that drives the transitions and structure-based mechanisms for its formation. Near-atomic resolution models of subunit tertiary structures in an early intermediate of bacteriophage HK97 maturation revealed a remarkable distortion of the secondary structures when compared to the mature particle. Scaffolding proteins may induce the distortion that is maintained by quaternary structure interactions following scaffold release, making the intermediate particle metastable.
James F. Conway - One of the best experts on this subject based on the ideXlab platform.
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Capsids and Genomes of Jumbo-Sized Bacteriophages Reveal the Evolutionary Reach of the HK97 Fold.
mBio, 2017Co-Authors: Jianfei Hua, Robert L. Duda, Roger W. Hendrix, Welkin H. Pope, Alexis Huet, Carlos A. Lopez, Katerina Toropova, James F. ConwayAbstract:Large icosahedral viruses that infect bacteria represent an extreme of the coevolution of capsids and the genomes they accommodate. One subset of these large viruses is the jumbophages, tailed phages with double-stranded DNA genomes of at least 200,000 bp. We explored the mechanism leading to increased capsid and genome sizes by characterizing structures of several jumbophage capsids and the DNA packaged within them. Capsid structures determined for six jumbophages were consistent with the canonical phage HK97 fold, and three had capsid geometries with novel triangulation numbers (T=25, T=28, and T=52). Packaged DNA (chromosome) sizes were larger than the genome sizes, indicating that all jumbophages use a head-full DNA packaging mechanism. For two phages (PAU and G), the sizes appeared very much larger than their genome length. We used two-dimensional DNA gel electrophoresis to show that these two DNAs migrated abnormally due to base modifications and to allow us to calculate their actual chromosome sizes. Our results support a ratchet model of capsid and genome coevolution whereby mutations lead to increased capsid volume and allow the acquisition of additional genes. Once the added genes and larger capsid are established, mutations that restore the smaller size are disfavored.IMPORTANCE A large family of viruses share the same fold of the capsid protein as bacteriophage HK97, a virus that infects bacteria. Members of this family use different numbers of the capsid protein to build capsids of different sizes. Here, we examined the structures of extremely large capsids and measured their DNA content relative to the sequenced genome lengths, aiming to understand the process that increases size. We concluded that mutational changes leading to larger capsids become locked in by subsequent changes to the genome organization.
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Correct Assembly of the Bacteriophage T5 Procapsid Requires Both the Maturation Protease and the Portal Complex
Journal of Molecular Biology, 2016Co-Authors: Alexis Huet, Robert L. Duda, Roger W. Hendrix, Pascale Boulanger, James F. ConwayAbstract:The 90-nm-diameter capsid of coliphage T5 is organized with T=13 icosahedral geometry and encloses a double-stranded DNA genome that measures 121kbp. Its assembly follows a path similar to that of phage HK97 but yielding a larger structure that includes 775 subunits of the major head protein, 12 subunits of the portal protein and 120 subunits of the decoration protein. As for phage HK97, T5 encodes the scaffold function as an N-terminal extension (∆-domain) to the major head protein that is cleaved by the maturation protease after assembly of the initial prohead I form and prior to DNA packaging and capsid expansion. Although the major head protein alone is sufficient to assemble capsid-like particles, the yield is poor and includes many deformed structures. Here we explore the role of both the portal and the protease in capsid assembly by generating constructs that include the major head protein and a combination of protease (wild type or an inactive mutant) and portal proteins and overexpressing them in Escherichia coli. Our results show that the inactive protease mutant acts to trigger assembly of the major head protein, probably through binding to the ∆-domain, while the portal protein regulates assembly into the correct T=13 geometry. A cryo-electron microscopy reconstruction of prohead I including inactivated protease reveals density projecting from the prohead interior surface toward its center that is compatible with the ∆-domain, as well as additional internal density that we assign as the inactivated protease. These results reveal complexity in T5 beyond that of the HK97 system.
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Metastable Intermediates as Stepping Stones on the Maturation Pathways of Viral Capsids
mBio, 2014Co-Authors: Giovanni Cardone, Robert L. Duda, Roger W. Hendrix, James F. Conway, Naiqian Cheng, Lili You, Alasdair C. StevenAbstract:As they mature, many capsids undergo massive conformational changes that transform their stability, reactivity, and capacity for DNA. In some cases, maturation proceeds via one or more intermediate states. These structures represent local min- ima in a rich energy landscape that combines contributions from subunit folding, association of subunits into capsomers, and intercapsomer interactions. We have used scanning calorimetry and cryo-electron microscopy to explore the range of capsid conformations accessible to bacteriophage HK97. To separate conformational effects from those associated with covalent cross- linking (a stabilization mechanism of HK97), a cross-link-incompetent mutant was used. The mature capsid Head I undergoes an endothermic phase transition at 60°C in which it shrinks by 7%, primarily through changes in its hexamer conformation. The transition is reversible, with a half-life of ~3 min; however, >50% of reverted capsids are severely distorted or ruptured. This observation implies that such damage is a potential hazard of large-scale structural changes such as those involved in matura- tion. Assuming that the risk is lower for smaller changes, this suggests a rationalization for the existence of metastable interme- diates: that they serve as stepping stones that preserve capsid integrity as it switches between the radically different conforma- tions of its precursor and mature states. IMPORTANCE Large-scale conformational changes are widespread in virus maturation and infection processes. These changes are accompanied by the release of conformational free energy as the virion (or fusogenic glycoprotein) switches from a precursor state to its mature state. Each state corresponds to a local minimum in an energy landscape. The conformational changes in cap- sid maturation are so radical that the question arises of how maturing capsids avoid being torn apart. Offering proof of princi- ple, severe damage is inflicted when a bacteriophage HK97 capsid reverts from the (nonphysiological) state that it enters when heated past 60°C. We suggest that capsid proteins have been selected in part by the criterion of being able to avoid sustaining collateral damage as they mature. One way of achieving this—as with the HK97 capsid—involves breaking the overall transition down into several smaller steps in which the risk of damage is reduced.
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The tripartite capsid gene of Salmonella phage Gifsy-2 yields a capsid assembly pathway engaging features from HK97 and λ
Virology, 2010Co-Authors: Gregory Effantin, Nara Figueroa-bossi, Guy Schoehn, Lionello Bossi, James F. ConwayAbstract:Phage Gifsy-2, a lambdoid phage infecting Salmonella, has an unusually large composite gene coding for its major capsid protein (mcp) at the C-terminal end, a ClpP-like protease at the N-terminus, and a {approx} 200 residue central domain of unknown function but which may have a scaffolding role. This combination of functions on a single coding region is more extensive than those observed in other phages such as HK97 (scaffold-capsid fusion) and {lambda} (protease-scaffold fusion). To study the structural phenotype of the unique Gifsy-2 capsid gene, we have purified Gifsy-2 particles and visualized capsids and procapsids by cryoelectron microscopy, determining structures to resolutions up to 12 A. The capsids have lambdoid T = 7 geometry and are well modeled with the atomic structures of HK97 mcp and phage {lambda} gpD decoration protein. Thus, the unique Gifsy-2 capsid protein gene yields a capsid maturation pathway engaging features from both phages HK97 and {lambda}.
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Structure and energetics of encapsidated DNA in bacteriophage HK97 studied by scanning calorimetry and cryo-electron microscopy.
Journal of molecular biology, 2009Co-Authors: Robert L. Duda, Roger W. Hendrix, James F. Conway, Naiqian Cheng, Brian Firek, Philip D. Ross, Alasdair C. StevenAbstract:Abstract Encapsidation of duplex DNA by bacteriophages represents an extreme case of genome condensation, reaching near-crystalline concentrations of DNA. The HK97 system is well suited to study this phenomenon in view of the detailed knowledge of its capsid structure. To characterize the interactions involved, we combined calorimetry with cryo-electron microscopy and native gel electrophoresis. We found that, as in other phages, HK97 DNA is organized in coaxially wound nested shells. When DNA-filled capsids (heads) are scanned in buffer containing 1 mM Mg 2+ , DNA melting and capsid denaturation both contribute to the complex thermal profile between 82 °C and 96 °C. In other conditions (absence of Mg 2+ and lower ionic strength), DNA melting shifts to lower temperatures and the two events are resolved. Heads release their DNA at temperatures well below the onset of DNA melting or capsid denaturation. We suggest that, on heating, the internal pressure increases, causing the DNA to exit—probably via the portal vertex–while the capsid, although largely intact, sustains local damage that leads to an earlier onset of thermal denaturation. Heads differ structurally from empty capsids in the curvature of their protein shell, a change attributable to outwards pressure exerted by the DNA. We propose that this transition is sensed by the portal that is embedded in the capsid wall, whereupon the structure of the portal and its interactions with terminase, the packaging enzyme, are altered, thus signaling that packaging is at or approaching completion.
Carolyn M Teschke - One of the best experts on this subject based on the ideXlab platform.
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A hydrophobic network: Intersubunit and intercapsomer interactions stabilizing the bacteriophage P22 capsid
2019Co-Authors: Kunica Asija, Carolyn M TeschkeAbstract:Abstract dsDNA tailed phages and herpesviruses assemble their capsids using coat proteins that have the ubiquitous HK97 fold. Though this fold is common, we do not have a thorough understanding of the different ways viruses adapt it to maintain stability in various environments. The HK97-fold E-loop, which connects adjacent subunits at the outer periphery of capsomers, has been implicated in capsid stability. Here we show that in bacteriophage P22, residue W61 at the tip of the E-loop plays a role in stabilizing procapsids and in maturation. We hypothesize that a hydrophobic pocket is formed by residues I366 and W410 in the P-domain of a neighboring subunit within a capsomer, into which W61 fits like a peg. In addition, W61 likely bridges to residues A91 and L401 in P-domain loops of an adjacent capsomer, thereby linking the entire capsid together with a network of hydrophobic interactions. There is conservation of this hydrophobic network in the distantly related P22-like phages, indicating that this structural feature is likely important for stabilizing this family of phages. Thus, our data shed light on one of the varied elegant mechanisms used in nature to consistently build stable viral genome containers through subtle adaptation of the HK97 fold. IMPORTANCE Similarities in assembly reactions and coat protein structures of the dsDNA tailed phages and herpesviruses make phages ideal models to understand capsid assembly and identify potential targets for antiviral drug discovery. The coat protein E-loops of these viruses are involved in both intra-and intercapsomer interactions. In phage P22, hydrophobic interactions peg the coat protein subunits together within a capsomer, where the E-loop hydrophobic residue W61 of one subunit packs into a pocket of hydrophobic residues I366 and W410 of the adjacent subunit. W61 also makes hydrophobic interactions with A91 and L401 of a subunit in an adjacent capsomer. We show these intra-and intercapsomer hydrophobic interactions form a network crucial to capsid stability and proper assembly.
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conservation and divergence of the i domain inserted into the ubiquitous HK97 coat protein fold in p22 like bacteriophages
Journal of Virology, 2019Co-Authors: Therese N. Tripler, Andrei T Alexandrescu, Anne R Kaplan, Carolyn M TeschkeAbstract:: Despite very low sequence homology, the major capsid proteins of double-stranded DNA (dsDNA) bacteriophages, some archaeal viruses, and the herpesviruses share a structural motif, the HK97 fold. Bacteriophage P22, a paradigm for this class of viruses, belongs to a phage gene cluster that contains three homology groups: P22-like, CUS-3-like, and Sf6-like. The coat protein of each phage has an inserted domain (I-domain) that is more conserved than the rest of the coat protein. In P22, loops in the I-domain are critical for stabilizing intra- and intersubunit contacts that guide proper capsid assembly. The nuclear magnetic resonance (NMR) structures of the P22, CUS-3, and Sf6 I-domains reveal that they are all six-stranded, anti-parallel β-barrels. Nevertheless, significant structural differences occur in loops connecting the β-strands, in surface electrostatics used to dock the I-domains with their respective coat protein core partners, and in sequence motifs displayed on the capsid surfaces. Our data highlight the structural diversity of I-domains that could lead to variations in capsid assembly mechanisms and capsid surfaces adapted for specific phage functions.IMPORTANCE Comparative studies of protein structures often provide insights into their evolution. The HK97 fold is a structural motif used to form the coat protein shells that encapsidate the genomes of many dsDNA phages and viruses. The structure and function of coat proteins based on the HK97 fold are often embellished by the incorporation of I-domains. In the present work we compare I-domains from three phages representative of highly divergent P22-like homology groups. While the three I-domains share a six-stranded β-barrel skeleton, there are differences (i) in structure elements at the periphery of the conserved fold, (ii) in the locations of disordered loops important in capsid assembly and conformational transitions, (iii) in surfaces charges, and (iv) in sequence motifs that are potential ligand-binding sites. These structural modifications on the rudimentary I-domain fold suggest that considerable structural adaptability was needed to fulfill the versatile range of functional requirements for distinct phages.
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The amazing HK97 fold: versatile results of modest differences
Current opinion in virology, 2019Co-Authors: Robert L. Duda, Carolyn M TeschkeAbstract:dsDNA Bacteriophages, some dsDNA archaeal viruses and the Herpesviruses share many features including a common capsid assembly pathway and coat protein fold. The coat proteins of these viruses, which have the HK97 fold, co-assemble with a free or attached scaffolding protein and other capsid proteins into a precursor capsid, known as a procapsid or prohead. The procapsid is a metastable state that increases in stability as a result of morphological changes that occur during the dsDNA packaging reaction. We review evidence from several systems indicating that proper contacts acquired in the assembly of the procapsid are critical to forming the correct morphology in the mature capsid.
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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
Journal of Virology, 2015Co-Authors: Nadia G Dlima, Carolyn M TeschkeAbstract:UNLABELLED: 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. IMPORTANCE: Many dsDNA viruses have morphogenic pathways utilizing an intermediate capsid, known as a procapsid. These procapsids are assembled from a coat protein having the HK97 fold in a reaction driven by scaffolding proteins or delta domains. Maturation of the capsid occurs during DNA packaging. Bacteriophage HK97 uniquely stabilizes its capsid during maturation by intercapsomer cross-linking, but most virus capsids are stabilized by alternate means. Here we show that the I domain that is inserted into the coat protein of bacteriophage P22 is important in the process of proper procapsid assembly. Specifically, the I domain allows for stabilizing interactions across the capsid 2-fold axis of symmetry via a D-loop. When amino acid residues at the tip of the D-loop are mutated, aberrant assembly products, including tubes, are formed instead of procapsids, consequently phage production is affected, indicating the importance of stabilizing interactions during the assembly and maturation reactions.
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Nature's favorite building block: Deciphering folding and capsid assembly of proteins with the HK97-fold.
Virology, 2015Co-Authors: Margaret M Suhanovsky, Carolyn M TeschkeAbstract:For many (if not all) bacterial and archaeal tailed viruses and eukaryotic Herpesvirdae the HK97-fold serves as the major architectural element in icosahedral capsid formation while still enabling the conformational flexibility required during assembly and maturation. Auxiliary proteins or Δ-domains strictly control assembly of multiple, identical, HK97-like subunits into procapsids with specific icosahedral symmetries, rather than aberrant non-icosahedral structures. Procapsids are precursor structures that mature into capsids in a process involving release of auxiliary proteins (or cleavage of Δ-domains), dsDNA packaging, and conformational rearrangement of the HK97-like subunits. Some coat proteins built on the ubiquitous HK97-fold also have accessory domains or loops that impart specific functions, such as increased monomer, procapsid, or capsid stability. In this review, we analyze the numerous HK97-like coat protein structures that are emerging in the literature (over 40 at time of writing) by comparing their topology, additional domains, and their assembly and misassembly reactions.