The cystovirus f6 shares several distinct features with other double-stranded RNA (dsRNA) viruses... more The cystovirus f6 shares several distinct features with other double-stranded RNA (dsRNA) viruses, including the human pathogen, rotavirus: segmented genomes, nonequivalent packing of 120 subunits in its icosahedral capsid, and capsids as compartments for transcription and replication. f6 assembles as a dodecahedral procapsid that undergoes major conformational changes as it matures into the spherical capsid. We determined the crystal structure of the capsid protein, P1, revealing a flattened trapezoid subunit with an a-helical fold. We also solved the procapsid with cryo-electron microscopy to comparable resolution. Fitting the crystal structure into the procapsid disclosed substantial conformational differences between the two P1 conformers. Maturation via two intermediate states involves remodeling on a similar scale, besides huge rigid-body rotations. The capsid structure and its stepwise maturation that is coupled to sequential packaging of three RNA segments sets the cystoviruses apart from other dsRNA viruses as a dynamic molecular machine.
ABSTRACT Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond,... more ABSTRACT Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009
The initial assembly product of bacteriophage ϕ6, the procapsid, undergoes major structural trans... more The initial assembly product of bacteriophage ϕ6, the procapsid, undergoes major structural transformation during the sequential packaging of its three segments of single-stranded RNA. The procapsid, a compact icosahedrally symmetric particle with deeply recessed vertices, expands to the spherical mature capsid, increasing the volume available to accommodate the genome by 2.5-fold. It has been proposed that expansion and packaging are linked, with each stage in expansion presenting a binding site for a particular RNA segment. To investigate procapsid transformability, we induced expansion by acidification, heating, and elevated salt concentration. Cryo-electron microscopy reconstructions after all three treatments yielded the same partially expanded particle. Analysis by cryo-electron tomography showed that all vertices of a given capsid were either in a compact or an expanded state, indicating a highly cooperative transition. To benchmark the mature capsid, we analyzed filled (in vivo packaged) capsids. When these particles were induced to release their RNA, they reverted to the same intermediate state as expanded procapsids (intermediate 1) or to a second, further expanded state (intermediate 2). This partial reversibility of expansion suggests that the mature spherical capsid conformation is obtained only when sufficient outward pressure is exerted by packaged RNA. The observation of two intermediates is consistent with the proposed threestep packaging process. The model is further supported by the observation that a mutant capable of packaging the second RNA segment without previously packaging the first segment has enhanced susceptibility for switching spontaneously from the procapsid to the first intermediate state.
Bacteriophage P22, a podovirus infecting strains of Salmonella typhimurium, packages a 42-kbp gen... more Bacteriophage P22, a podovirus infecting strains of Salmonella typhimurium, packages a 42-kbp genome using a headful mechanism. DNA translocation is accomplished by the phage terminase, a powerful molecular motor consisting of large and small subunits. Although many of the structural proteins of the P22 virion have been well characterized, little is known about the terminase subunits and their molecular mechanism of DNA translocation. We report here structural and assembly properties of ectopically expressed and highly purified terminase large and small subunits. The large subunit (gp2), which contains the nuclease and ATPase activities of terminase, exists as a stable monomer with an α/β fold. The small subunit (gp3), which recognizes DNA for packaging and may regulate gp2 activity, exhibits a highly α-helical secondary structure and self-associates to form a stable oligomeric ring in solution. For wild-type gp3, the ring contains nine subunits, as demonstrated by hydrodynamic measurements, electron microscopy, and native mass spectrometry. We have also characterized a gp3 mutant (Ala 112 → Thr) that forms a 10-subunit ring, despite a subunit fold indistinguishable from wild type. Both the nonameric and decameric gp3 rings exhibit nonspecific DNA-binding activity, and gp2 is able to bind strongly to the DNA/gp3 complex but not to DNA alone. We propose a scheme for the roles of P22 terminase large and small subunits in the recruitment and packaging of viral DNA and discuss the model in relation to proposals for terminase-driven DNA translocation in other phages.
An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the a... more An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2-103 (Δ-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Δ-domain is thought to resemble a phage scaffolding protein, by virtue of its highly α-helical secondary structure and a tertiary fold that projects inward from the interior surface of the shell. Here, we employ factor analysis of temperature-dependent Raman spectra to characterize the thermostability of the Δ-domain secondary structure and to quantify the thermodynamic parameters of Δ-domain unfolding. The results are compared for the Δ-domain within the prohead I architecture (in situ) and for a recombinantly expressed 111-residue peptide (in vitro). We find that the α-helicity (∼70%), median melting temperature (T m = 58°C), enthalpy (ΔH m = 50 ± 5 kcal mol − 1 ), entropy (ΔS m = 150 ± 10 cal mol − 1 K − 1 ), and average cooperative melting unit (〈n c 〉 ∼ 3.5) of the in situ Δ-domain are altered in vitro, indicating specific interdomain interactions within prohead I. Thus, the in vitro Δ-domain, despite an enhanced helical secondary structure (∼ 90% α-helix), exhibits diminished thermostability (T m = 40°C; ΔH m = 27 ± 2 kcal mol − 1 ; ΔS m = 86 ± 6 cal mol − 1 K − 1 ) and noncooperative unfolding (〈n c 〉 ∼ 1) vis-à-vis the in situ Δ-domain. Temperature-dependent Raman markers of subunit side chains, particularly those of Phe and Trp residues, also confirm different local interactions for the in situ and in vitro Δ-domains. The present results clarify the key role of the gp5 Δ-domain in prohead I architecture by providing direct evidence of domain structure stabilization and interdomain interactions within the assembled shell.
The structures of complex biological assemblies, such as viruses, and the molecular mechanisms go... more The structures of complex biological assemblies, such as viruses, and the molecular mechanisms governing virus assembly in vivo and in vitro require investigation by multiple biophysical techniques. One such technique is Raman spectroscopy, which is particularly useful for probing key steps in the assembly pathways of viruses and for elucidating the molecular structures of viral proteins and nucleic acids. Here, we briefly review the implementation of methods of Raman spectroscopy, including ultraviolet resonance Raman (UVRR) spectroscopy and polarized Raman spectroscopy as structural probes of native viruses, viral precursor assemblies and their constituent proteins, and nucleic acids. We illustrate the powerful analytical approaches of singular value decomposition (SVD) and polarized Raman microspectroscopy for unique quantitative assessments of viral protein structures. Attention is focused on recent applications to bacterial viruses (bacteriophages) of icosahedral (P22 and HK97)...
Bacteriophage ϕ6 is a double-stranded RNA virus that has been extensively studied as a model orga... more Bacteriophage ϕ6 is a double-stranded RNA virus that has been extensively studied as a model organism. Here we describe structure determination of ϕ6 major capsid protein P1. The protein crystallized in base centered orthorhombic space group C2221. Matthews's coefficient indicated that the crystals contain from four to seven P1 subunits in the crystallographic asymmetric unit. The self-rotation function had shown presence of fivefold axes of non-crystallographic symmetry in the crystals. Thus, electron density map corresponding to a P1 pentamer was excised from a previously determined cryoEM reconstruction of the ϕ6 procapsid at 7 Å resolution and used as a model for molecular replacement. The phases for reflections at higher than 7 Å resolution were obtained by phase extension employing the fivefold non-crystallographic symmetry present in the crystal. The averaged 3.6 Å-resolution electron density map was of sufficient quality to allow model building.
Living cells compartmentalize materials and enzymatic reactions to increase metabolic efficiency.... more Living cells compartmentalize materials and enzymatic reactions to increase metabolic efficiency. While eukaryotes use membranebound organelles, bacteria and archaea rely primarily on proteinbound nanocompartments. Encapsulins constitute a class of nanocompartments widespread in bacteria and archaea whose functions have hitherto been unclear. Here, we characterize the encapsulin nanocompartment from Myxococcus xanthus, which consists of a shell protein (EncA, 32.5 kDa) and three internal proteins (EncB, 17 kDa; EncC, 13 kDa; EncD, 11 kDa). Using cryo-electron microscopy, we determined that EncA self-assembles into an icosahedral shell 32 nm in diameter (26 nm internal diameter), built from 180 subunits with the fold first observed in bacteriophage HK97 capsid. The internal proteins, of which EncB and EncC have ferritin-like domains, attach to its inner surface. Native nanocompartments have dense iron-rich cores. Functionally, they resemble ferritins, cage-like iron storage proteins, but with a massively greater capacity (~30,000 iron atoms versus~3,000 in ferritin). Physiological data reveal that few nanocompartments are assembled during vegetative growth, but they increase fivefold upon starvation, protecting cells from oxidative stress through iron sequestration.
Iron is both an essential cofactor of many enzymes and a producer of highly reactive hydroxyl rad... more Iron is both an essential cofactor of many enzymes and a producer of highly reactive hydroxyl radicals that can cause cellular damage. To regulate the supply of intracellular iron, cells have developed protein-based organelles, like ferritins, that act as iron storage containers. Myxococcus xanthus, a soil-dwelling gram-negative myxobacterium, produces another type of protein-based organelle that has been related to iron metabolism, the encapsulin nanocompartment [1]. It has a spherical shell composed of a 32 kDa protein called EncA or encapsulin, and three minor proteins in the 11 kDa to 17 kDa range . To gain insight on the molecular architecture of this complex, we have studied encapsulin nanocompartments purified from M. xanthus and recombinant EncA shells produced in E. coli using single-particle cryo-electron microscopy (cryo-EM). This analysis was supplemented with conventional TEM with and without negative staining and scanning transmission electron microscopy (STEM).
Current protocols in protein science / editorial board, John E. Coligan ... [et al.], 2013
A protein Raman spectrum comprises discrete bands representing vibrational modes of the peptide b... more A protein Raman spectrum comprises discrete bands representing vibrational modes of the peptide backbone and its side chains. The spectral positions, intensities, and polarizations of the Raman bands are sensitive to protein secondary, tertiary, and quaternary structures and to side-chain orientations and local environments. In favorable cases, the Raman spectrum serves as an empirical signature of protein three-dimensional structure, intramolecular dynamics, and intermolecular interactions. Quantitative analysis of Raman spectral series can be further boosted by advanced statistical approaches of factor analysis that allow fitting of specific theoretical models while reducing the amount of analyzed data. Here, the strengths of Raman spectroscopy are illustrated by considering recent applications from the authors' work that address (1) subunit folding and recognition in assembly of the icosahedral bacteriophages, (2) orientations of subunit main chains and side chains in native ...
The cystovirus f6 shares several distinct features with other double-stranded RNA (dsRNA) viruses... more The cystovirus f6 shares several distinct features with other double-stranded RNA (dsRNA) viruses, including the human pathogen, rotavirus: segmented genomes, nonequivalent packing of 120 subunits in its icosahedral capsid, and capsids as compartments for transcription and replication. f6 assembles as a dodecahedral procapsid that undergoes major conformational changes as it matures into the spherical capsid. We determined the crystal structure of the capsid protein, P1, revealing a flattened trapezoid subunit with an a-helical fold. We also solved the procapsid with cryo-electron microscopy to comparable resolution. Fitting the crystal structure into the procapsid disclosed substantial conformational differences between the two P1 conformers. Maturation via two intermediate states involves remodeling on a similar scale, besides huge rigid-body rotations. The capsid structure and its stepwise maturation that is coupled to sequential packaging of three RNA segments sets the cystoviruses apart from other dsRNA viruses as a dynamic molecular machine.
The cystovirus f6 shares several distinct features with other double-stranded RNA (dsRNA) viruses... more The cystovirus f6 shares several distinct features with other double-stranded RNA (dsRNA) viruses, including the human pathogen, rotavirus: segmented genomes, nonequivalent packing of 120 subunits in its icosahedral capsid, and capsids as compartments for transcription and replication. f6 assembles as a dodecahedral procapsid that undergoes major conformational changes as it matures into the spherical capsid. We determined the crystal structure of the capsid protein, P1, revealing a flattened trapezoid subunit with an a-helical fold. We also solved the procapsid with cryo-electron microscopy to comparable resolution. Fitting the crystal structure into the procapsid disclosed substantial conformational differences between the two P1 conformers. Maturation via two intermediate states involves remodeling on a similar scale, besides huge rigid-body rotations. The capsid structure and its stepwise maturation that is coupled to sequential packaging of three RNA segments sets the cystoviruses apart from other dsRNA viruses as a dynamic molecular machine.
ABSTRACT Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond,... more ABSTRACT Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009
The initial assembly product of bacteriophage ϕ6, the procapsid, undergoes major structural trans... more The initial assembly product of bacteriophage ϕ6, the procapsid, undergoes major structural transformation during the sequential packaging of its three segments of single-stranded RNA. The procapsid, a compact icosahedrally symmetric particle with deeply recessed vertices, expands to the spherical mature capsid, increasing the volume available to accommodate the genome by 2.5-fold. It has been proposed that expansion and packaging are linked, with each stage in expansion presenting a binding site for a particular RNA segment. To investigate procapsid transformability, we induced expansion by acidification, heating, and elevated salt concentration. Cryo-electron microscopy reconstructions after all three treatments yielded the same partially expanded particle. Analysis by cryo-electron tomography showed that all vertices of a given capsid were either in a compact or an expanded state, indicating a highly cooperative transition. To benchmark the mature capsid, we analyzed filled (in vivo packaged) capsids. When these particles were induced to release their RNA, they reverted to the same intermediate state as expanded procapsids (intermediate 1) or to a second, further expanded state (intermediate 2). This partial reversibility of expansion suggests that the mature spherical capsid conformation is obtained only when sufficient outward pressure is exerted by packaged RNA. The observation of two intermediates is consistent with the proposed threestep packaging process. The model is further supported by the observation that a mutant capable of packaging the second RNA segment without previously packaging the first segment has enhanced susceptibility for switching spontaneously from the procapsid to the first intermediate state.
Bacteriophage P22, a podovirus infecting strains of Salmonella typhimurium, packages a 42-kbp gen... more Bacteriophage P22, a podovirus infecting strains of Salmonella typhimurium, packages a 42-kbp genome using a headful mechanism. DNA translocation is accomplished by the phage terminase, a powerful molecular motor consisting of large and small subunits. Although many of the structural proteins of the P22 virion have been well characterized, little is known about the terminase subunits and their molecular mechanism of DNA translocation. We report here structural and assembly properties of ectopically expressed and highly purified terminase large and small subunits. The large subunit (gp2), which contains the nuclease and ATPase activities of terminase, exists as a stable monomer with an α/β fold. The small subunit (gp3), which recognizes DNA for packaging and may regulate gp2 activity, exhibits a highly α-helical secondary structure and self-associates to form a stable oligomeric ring in solution. For wild-type gp3, the ring contains nine subunits, as demonstrated by hydrodynamic measurements, electron microscopy, and native mass spectrometry. We have also characterized a gp3 mutant (Ala 112 → Thr) that forms a 10-subunit ring, despite a subunit fold indistinguishable from wild type. Both the nonameric and decameric gp3 rings exhibit nonspecific DNA-binding activity, and gp2 is able to bind strongly to the DNA/gp3 complex but not to DNA alone. We propose a scheme for the roles of P22 terminase large and small subunits in the recruitment and packaging of viral DNA and discuss the model in relation to proposals for terminase-driven DNA translocation in other phages.
An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the a... more An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2-103 (Δ-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Δ-domain is thought to resemble a phage scaffolding protein, by virtue of its highly α-helical secondary structure and a tertiary fold that projects inward from the interior surface of the shell. Here, we employ factor analysis of temperature-dependent Raman spectra to characterize the thermostability of the Δ-domain secondary structure and to quantify the thermodynamic parameters of Δ-domain unfolding. The results are compared for the Δ-domain within the prohead I architecture (in situ) and for a recombinantly expressed 111-residue peptide (in vitro). We find that the α-helicity (∼70%), median melting temperature (T m = 58°C), enthalpy (ΔH m = 50 ± 5 kcal mol − 1 ), entropy (ΔS m = 150 ± 10 cal mol − 1 K − 1 ), and average cooperative melting unit (〈n c 〉 ∼ 3.5) of the in situ Δ-domain are altered in vitro, indicating specific interdomain interactions within prohead I. Thus, the in vitro Δ-domain, despite an enhanced helical secondary structure (∼ 90% α-helix), exhibits diminished thermostability (T m = 40°C; ΔH m = 27 ± 2 kcal mol − 1 ; ΔS m = 86 ± 6 cal mol − 1 K − 1 ) and noncooperative unfolding (〈n c 〉 ∼ 1) vis-à-vis the in situ Δ-domain. Temperature-dependent Raman markers of subunit side chains, particularly those of Phe and Trp residues, also confirm different local interactions for the in situ and in vitro Δ-domains. The present results clarify the key role of the gp5 Δ-domain in prohead I architecture by providing direct evidence of domain structure stabilization and interdomain interactions within the assembled shell.
The structures of complex biological assemblies, such as viruses, and the molecular mechanisms go... more The structures of complex biological assemblies, such as viruses, and the molecular mechanisms governing virus assembly in vivo and in vitro require investigation by multiple biophysical techniques. One such technique is Raman spectroscopy, which is particularly useful for probing key steps in the assembly pathways of viruses and for elucidating the molecular structures of viral proteins and nucleic acids. Here, we briefly review the implementation of methods of Raman spectroscopy, including ultraviolet resonance Raman (UVRR) spectroscopy and polarized Raman spectroscopy as structural probes of native viruses, viral precursor assemblies and their constituent proteins, and nucleic acids. We illustrate the powerful analytical approaches of singular value decomposition (SVD) and polarized Raman microspectroscopy for unique quantitative assessments of viral protein structures. Attention is focused on recent applications to bacterial viruses (bacteriophages) of icosahedral (P22 and HK97)...
Bacteriophage ϕ6 is a double-stranded RNA virus that has been extensively studied as a model orga... more Bacteriophage ϕ6 is a double-stranded RNA virus that has been extensively studied as a model organism. Here we describe structure determination of ϕ6 major capsid protein P1. The protein crystallized in base centered orthorhombic space group C2221. Matthews's coefficient indicated that the crystals contain from four to seven P1 subunits in the crystallographic asymmetric unit. The self-rotation function had shown presence of fivefold axes of non-crystallographic symmetry in the crystals. Thus, electron density map corresponding to a P1 pentamer was excised from a previously determined cryoEM reconstruction of the ϕ6 procapsid at 7 Å resolution and used as a model for molecular replacement. The phases for reflections at higher than 7 Å resolution were obtained by phase extension employing the fivefold non-crystallographic symmetry present in the crystal. The averaged 3.6 Å-resolution electron density map was of sufficient quality to allow model building.
Living cells compartmentalize materials and enzymatic reactions to increase metabolic efficiency.... more Living cells compartmentalize materials and enzymatic reactions to increase metabolic efficiency. While eukaryotes use membranebound organelles, bacteria and archaea rely primarily on proteinbound nanocompartments. Encapsulins constitute a class of nanocompartments widespread in bacteria and archaea whose functions have hitherto been unclear. Here, we characterize the encapsulin nanocompartment from Myxococcus xanthus, which consists of a shell protein (EncA, 32.5 kDa) and three internal proteins (EncB, 17 kDa; EncC, 13 kDa; EncD, 11 kDa). Using cryo-electron microscopy, we determined that EncA self-assembles into an icosahedral shell 32 nm in diameter (26 nm internal diameter), built from 180 subunits with the fold first observed in bacteriophage HK97 capsid. The internal proteins, of which EncB and EncC have ferritin-like domains, attach to its inner surface. Native nanocompartments have dense iron-rich cores. Functionally, they resemble ferritins, cage-like iron storage proteins, but with a massively greater capacity (~30,000 iron atoms versus~3,000 in ferritin). Physiological data reveal that few nanocompartments are assembled during vegetative growth, but they increase fivefold upon starvation, protecting cells from oxidative stress through iron sequestration.
Iron is both an essential cofactor of many enzymes and a producer of highly reactive hydroxyl rad... more Iron is both an essential cofactor of many enzymes and a producer of highly reactive hydroxyl radicals that can cause cellular damage. To regulate the supply of intracellular iron, cells have developed protein-based organelles, like ferritins, that act as iron storage containers. Myxococcus xanthus, a soil-dwelling gram-negative myxobacterium, produces another type of protein-based organelle that has been related to iron metabolism, the encapsulin nanocompartment [1]. It has a spherical shell composed of a 32 kDa protein called EncA or encapsulin, and three minor proteins in the 11 kDa to 17 kDa range . To gain insight on the molecular architecture of this complex, we have studied encapsulin nanocompartments purified from M. xanthus and recombinant EncA shells produced in E. coli using single-particle cryo-electron microscopy (cryo-EM). This analysis was supplemented with conventional TEM with and without negative staining and scanning transmission electron microscopy (STEM).
Current protocols in protein science / editorial board, John E. Coligan ... [et al.], 2013
A protein Raman spectrum comprises discrete bands representing vibrational modes of the peptide b... more A protein Raman spectrum comprises discrete bands representing vibrational modes of the peptide backbone and its side chains. The spectral positions, intensities, and polarizations of the Raman bands are sensitive to protein secondary, tertiary, and quaternary structures and to side-chain orientations and local environments. In favorable cases, the Raman spectrum serves as an empirical signature of protein three-dimensional structure, intramolecular dynamics, and intermolecular interactions. Quantitative analysis of Raman spectral series can be further boosted by advanced statistical approaches of factor analysis that allow fitting of specific theoretical models while reducing the amount of analyzed data. Here, the strengths of Raman spectroscopy are illustrated by considering recent applications from the authors' work that address (1) subunit folding and recognition in assembly of the icosahedral bacteriophages, (2) orientations of subunit main chains and side chains in native ...
The cystovirus f6 shares several distinct features with other double-stranded RNA (dsRNA) viruses... more The cystovirus f6 shares several distinct features with other double-stranded RNA (dsRNA) viruses, including the human pathogen, rotavirus: segmented genomes, nonequivalent packing of 120 subunits in its icosahedral capsid, and capsids as compartments for transcription and replication. f6 assembles as a dodecahedral procapsid that undergoes major conformational changes as it matures into the spherical capsid. We determined the crystal structure of the capsid protein, P1, revealing a flattened trapezoid subunit with an a-helical fold. We also solved the procapsid with cryo-electron microscopy to comparable resolution. Fitting the crystal structure into the procapsid disclosed substantial conformational differences between the two P1 conformers. Maturation via two intermediate states involves remodeling on a similar scale, besides huge rigid-body rotations. The capsid structure and its stepwise maturation that is coupled to sequential packaging of three RNA segments sets the cystoviruses apart from other dsRNA viruses as a dynamic molecular machine.
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Papers by Daniel Nemecek