RESUMEN
Molecular communication across physical barriers requires pores to connect the environments on either side and discriminate between the diffusants. Here we use porous virus-like particles (VLPs) derived from bacteriophage P22 to investigate the range of molecule sizes able to gain access to its interior. Although there are cryo-EM models of the VLP, they may not accurately depict the parameters of the molecules able to pass across the pores due to the dynamic nature of the P22 particles in the solution. After encapsulating the enzyme AdhD within the P22 VLPs, we use a redox reaction involving PAMAM dendrimer modified NADH/NAD+ to examine the size and charge limitations of molecules entering P22. Utilizing the three different accessible morphologies of the P22 particles, we determine the effective pore sizes of each and demonstrate that negatively charged substrates diffuse across more readily when compared to those that are neutral, despite the negatively charge exterior of the particles.
Asunto(s)
Bacteriófago P22/metabolismo , Proteínas de la Cápside/metabolismo , Cápside/metabolismo , Virión/metabolismo , Algoritmos , Bacteriófago P22/genética , Bacteriófago P22/ultraestructura , Cápside/ultraestructura , Proteínas de la Cápside/genética , Microscopía por Crioelectrón , Dendrímeros/química , Dendrímeros/metabolismo , Difusión , Microscopía Electrónica de Transmisión , Modelos Teóricos , Mutación , NAD/química , NAD/metabolismo , Tamaño de la Partícula , Porosidad , Electricidad Estática , Virión/genética , Virión/ultraestructuraRESUMEN
Tailed dsDNA bacteriophages and herpesviruses form capsids using coat proteins that have the HK97 fold. In these viruses, the coat proteins first assemble into procapsids, which subsequently mature during DNA packaging. Generally interactions between the coat protein E-loop of one subunit and the P-domain of an adjacent subunit help stabilize both capsomers and capsids. Based on a recent 3.3â¯Å cryo-EM structure of the bacteriophage P22 virion, E-loop amino acids E52, E59 and E72 were suggested to stabilize the capsid through intra-capsomer salt bridges with the P-domain residues R102, R109 and K118. The glutamic acid residues were each mutated to alanine to test this hypothesis. The substitutions resulted in a WT phenotype and did not destabilize capsids; rather, the alanine substituted coat proteins increased the stability of procapsids and virions. These results indicate that different types of interactions must be used between the E-loop and P-domain to stabilize phage P22 procapsids and virions.
Asunto(s)
Bacteriófago P22/metabolismo , Proteínas de la Cápside/química , Cápside/química , Bacteriófago P22/química , Bacteriófago P22/genética , Bacteriófago P22/ultraestructura , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Modelos Moleculares , Dominios Proteicos , Estabilidad Proteica , Virión/química , Virión/genética , Virión/metabolismoRESUMEN
For successful infection, bacteriophages must overcome multiple barriers to transport their genome and proteins across the bacterial cell envelope. We use cryo-electron tomography to study the infection initiation of phage P22 in Salmonella enterica serovar Typhimurium, revealing how a channel forms to allow genome translocation into the cytoplasm. Our results show free phages that initially attach obliquely to the cell through interactions between the O antigen and two of the six tailspikes; the tail needle also abuts the cell surface. The virion then orients perpendicularly and the needle penetrates the outer membrane. The needle is released and the internal head protein gp7* is ejected and assembles into an extracellular channel that extends from the gp10 baseplate to the cell surface. A second protein, gp20, is ejected and assembles into a structure that extends the extracellular channel across the outer membrane into the periplasm. Insertion of the third ejected protein, gp16, into the cytoplasmic membrane probably completes the overall trans-envelope channel into the cytoplasm. Construction of a trans-envelope channel is an essential step during infection of Gram-negative bacteria by all short-tailed phages, because such virions cannot directly deliver their genome into the cell cytoplasm.
Asunto(s)
Bacteriófago P22/fisiología , Membrana Celular/metabolismo , Membrana Celular/virología , Tomografía con Microscopio Electrónico/métodos , Salmonella typhimurium/virología , Acoplamiento Viral , Internalización del Virus , Bacteriófago P22/patogenicidad , Bacteriófago P22/ultraestructura , Membrana Celular/ultraestructura , Citoplasma/metabolismo , Citoplasma/virología , ADN Viral , Modelos Moleculares , Antígenos O , Conformación Proteica , Proteínas de la Cola de los Virus/química , Virión/metabolismoRESUMEN
Virus-like particles (VLPs) resemble viruses, but are devoid their genetic material, rendering them as noninfectious, hollow protein shells. VLPs are ideal templates to synthesize nanoparticles because they have homogeneous size and their empty cavity can provide a confined environment for selectively directed synthesis. Atom-transfer radical polymerization (ATRP) is well suited for directed synthesis of polymers inside VLPs. In addition to being rapid, monomer-promiscuous, and resulting in products with relatively low polydispersity, the simplicity of the ATRP initiator allows it to be readily modified for amending to biomolecules. This chapter describes the polymerization of 2-aminoethyl methacrylate (AEMA) via ATRP in a viral capsid derived from the bacteriophage P22.
Asunto(s)
Bacteriófago P22 , Proteínas de la Cápside , Cápside , Nanocápsulas , Bacteriófago P22/química , Bacteriófago P22/metabolismo , Bacteriófago P22/ultraestructura , Cápside/química , Cápside/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Cromatografía Líquida de Alta Presión , Clonación Molecular , Reactivos de Enlaces Cruzados , Expresión Génica , Nanocápsulas/química , Nanocápsulas/ultraestructura , Multimerización de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Ensamble de VirusRESUMEN
Genome ejection proteins are required to facilitate transport of bacteriophage P22 double-stranded DNA safely through membranes of Salmonella. The structures and locations of all proteins in the context of the mature virion are known, with the exception of three ejection proteins. Furthermore, the changes that occur to the proteins residing in the mature virion upon DNA release are not fully understood. We used cryogenic electron microscopy to obtain what is, to our knowledge, the first asymmetric reconstruction of mature bacteriophage P22 after double-stranded DNA has been extruded from the capsid-a state representative of one step during viral infection. Results of icosahedral and asymmetric reconstructions at estimated resolutions of 7.8 and 12.5 Å resolutions, respectively, are presented. The reconstruction shows tube-like protein density extending from the center of the tail assembly. The portal protein does not revert to the more contracted, procapsid state, but instead maintains an extended and splayed barrel structure. These structural details contribute to our understanding of the molecular mechanism of P22 phage infection and also set the foundation for future exploitation serving engineering purposes.
Asunto(s)
Bacteriófago P22/genética , Bacteriófago P22/ultraestructura , Microscopía por Crioelectrón , Genoma Viral/genética , Virión/genética , Virión/ultraestructura , ADN Viral/metabolismoRESUMEN
Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or 'procapsid') built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of 'Headful Packaging' is a DNA-dependent symmetrization of portal protein.
Asunto(s)
Bacteriófago P22/fisiología , Proteínas de la Cápside/química , Cápside/fisiología , ADN Viral/fisiología , Ensamble de Virus/fisiología , Bacteriófago P22/ultraestructura , Cápside/ultraestructura , Proteínas de la Cápside/fisiología , Proteínas de la Cápside/ultraestructura , Cristalografía por Rayos X , Empaquetamiento del ADN/fisiología , ADN Viral/ultraestructura , Endodesoxirribonucleasas/metabolismo , Genoma Viral/fisiología , Microscopía Electrónica , Simulación del Acoplamiento Molecular , Multimerización de Proteína/fisiología , Estructura Cuaternaria de Proteína/fisiologíaRESUMEN
UNLABELLED: The P22 capsid is a T=7 icosahedrally symmetric protein shell with a portal protein dodecamer at one 5-fold vertex. Extending outwards from that vertex is a short tail, and putatively extending inwards is a 15-nm-long α-helical barrel formed by the C-terminal domains of portal protein subunits. In addition to the densely packed genome, the capsid contains three "ejection proteins" (E-proteins [gp7, gp16, and gp20]) destined to exit from the tightly sealed capsid during the process of DNA delivery into target cells. We estimated their copy numbers by quantitative SDS-PAGE as approximately 12 molecules per virion of gp16 and gp7 and 30 copies of gp20. To localize them, we used bubblegram imaging, an adaptation of cryo-electron microscopy in which gaseous bubbles induced in proteins by prolonged irradiation are used to map the proteins' locations. We applied this technique to wild-type P22, a triple mutant lacking all three E-proteins, and three mutants each lacking one E-protein. We conclude that all three E-proteins are loosely clustered around the portal axis, in the region displaced radially inwards from the portal crown. The bubblegram data imply that approximately half of the α-helical barrel seen in the portal crystal structure is disordered in the mature virion, and parts of the disordered region present binding sites for E-proteins. Thus positioned, the E-proteins are strategically placed to pass down the shortened barrel and through the portal ring and the tail, as they exit from the capsid during an infection. IMPORTANCE: While it has long been appreciated that capsids serve as delivery vehicles for viral genomes, there is now growing awareness that viruses also deliver proteins into their host cells. P22 has three such proteins (ejection proteins [E-proteins]), whose initial locations in the virion have remained unknown despite their copious amounts (total of 2.5 MDa). This study succeeded in localizing them by the novel technique of bubblegram imaging. The P22 E-proteins are seen to be distributed around the orifice of the portal barrel. Interestingly, this barrel, 15 nm long in a crystal structure, is only about half as long in situ: the remaining, disordered, portion appears to present binding sites for E-proteins. These observations document a spectacular example of a regulatory order-disorder transition in a supramolecular system and demonstrate the potential of bubblegram imaging to map the components of other viruses as well as cellular complexes.
Asunto(s)
Bacteriófago P22/química , Microscopía por Crioelectrón , Proteínas Virales/análisis , Virión/química , Bacteriófago P22/ultraestructura , Modelos Biológicos , Virión/ultraestructuraRESUMEN
Virus-like particles (VLPs) are well established platforms for constructing functional biomimetic materials. The VLP from the bacteriophage P22 can be used as a nanocontainer to sequester active enzymes, at high concentration, within its cavity through a process of directed self-assembly. Construction of ordered 2D assemblies of these catalytic VLPs can be envisioned as a functional membrane. To achieve this, it is important to establish methods to fabricate densely packed monolayers of VLPs. Highly ordered assemblies of P22 can also be utilized as a two-dimensional (2D) crystal for electron crystallography to get precise structural information on the VLP. Here we report 2D crystallization of different P22 morphologies: P22 procapsid (PC), enzyme encapsulated PC (ß-glycosidase and enhanced green fluorescent protein), empty shell (PC without scaffold proteins, ES), the expanded form of P22 (EX), and enzyme encapsulated EX (NADH oxidase). The 2D crystals of P22 VLPs were formed on a positively charged lipid monolayer at the water-air interface with a subphase containing 1% trehalose. A P22 solution, injected underneath the lipid monolayer, floated to the surface because of the density difference between the subphase and protein solution. The lipid monolayer, with adsorbed P22, was transferred to a holey carbon grid and was examined by electron microscopy. 2D crystals were obtained from a subphase containing 100 mM NaCl, 10 mM MES (pH 5.0), and 1% trehalose. The diffraction spots from the transferred film extended to the sixth order in negatively stained samples and the 10th order in cryo-electron microscopy samples.
Asunto(s)
Bacteriófago P22/química , Materiales Biomiméticos/química , Cristalización/métodos , Virión/química , Aire/análisis , Bacteriófago P22/ultraestructura , Microscopía por Crioelectrón , Dimiristoilfosfatidilcolina/química , Composición de Medicamentos , Proteínas Fluorescentes Verdes/química , Complejos Multienzimáticos/química , Miristatos/química , NADH NADPH Oxidorreductasas/química , Compuestos de Amonio Cuaternario/química , Electricidad Estática , Propiedades de Superficie , Trehalosa/química , Virión/ultraestructura , Agua/química , beta-Glucosidasa/químicaRESUMEN
CryoEM continues to produce density maps of larger and more complex assemblies with multiple protein components of mixed symmetries. Resolution is not always uniform throughout a cryoEM map, and it can be useful to estimate the resolution in specific molecular components of a large assembly. In this study, we present procedures to 1) estimate the resolution in subcomponents by gold-standard Fourier shell correlation (FSC); 2) validate modeling procedures, particularly at medium resolutions, which can include loop modeling and flexible fitting; and 3) build probabilistic models that combine high-accuracy priors (such as crystallographic structures) with medium-resolution cryoEM densities. As an example, we apply these methods to new cryoEM maps of the mature bacteriophage P22, reconstructed without imposing icosahedral symmetry. Resolution estimates based on gold-standard FSC show the highest resolution in the coat region (7.6 Å), whereas other components are at slightly lower resolutions: portal (9.2 Å), hub (8.5 Å), tailspike (10.9 Å), and needle (10.5 Å). These differences are indicative of inherent structural heterogeneity and/or reconstruction accuracy in different subcomponents of the map. Probabilistic models for these subcomponents provide new insights, to our knowledge, and structural information when taking into account uncertainty given the limitations of the observed density.
Asunto(s)
Bacteriófago P22/ultraestructura , Microscopía por Crioelectrón/métodos , Modelos Estadísticos , Bacteriófago P22/química , Proteínas de la Cápside/química , Modelos Moleculares , Conformación Proteica , Salmonella typhimurium/virologíaRESUMEN
Bacteriophage P22 has long been considered a hallmark model for virus assembly and maturation. Repurposing of P22 and other similar virus structures for nanotechnology and nanomedicine has reinvigorated the need to further understand the protein-protein interactions that allow for the assembly, as well as the conformational shifts required for maturation. In this work, gp5, the major coat structural protein of P22, has been manipulated in order to examine the mutational effects on procapsid stability and maturation. Insertions to the P22 coat protein A-domain, while widely permissive of procapsid assembly, destabilize the interactions necessary for virus maturation and potentially allow for the tunable adjustment of procapsid stability. Future manipulation of this region of the coat protein subunit can potentially be used to alter the stability of the capsid for controllable disassembly.
Asunto(s)
Bacteriófago P22/genética , Proteínas de la Cápside/química , Cápside/metabolismo , Regulación Viral de la Expresión Génica , Bacteriófago P22/metabolismo , Bacteriófago P22/ultraestructura , Secuencia de Bases , Cápside/ultraestructura , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Escherichia coli/virología , Calor , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Ingeniería de Proteínas , Pliegue de Proteína , Estructura Terciaria de Proteína , Ensamble de VirusRESUMEN
Some capsid proteins built on the ubiquitous HK97-fold have accessory domains imparting specific functions. Bacteriophage P22 coat protein has a unique insertion domain (I-domain). Two prior I-domain models from subnanometer cryoelectron microscopy (cryoEM) reconstructions differed substantially. Therefore, the I-domain's nuclear magnetic resonance structure was determined and also used to improve cryoEM models of coat protein. The I-domain has an antiparallel six-stranded ß-barrel fold, not previously observed in HK97-fold accessory domains. The D-loop, which is dynamic in the isolated I-domain and intact monomeric coat protein, forms stabilizing salt bridges between adjacent capsomers in procapsids. The S-loop is important for capsid size determination, likely through intrasubunit interactions. Ten of 18 coat protein temperature-sensitive-folding substitutions are in the I-domain, indicating its importance in folding and stability. Several are found on a positively charged face of the ß-barrel that anchors the I-domain to a negatively charged surface of the coat protein HK97-core.
Asunto(s)
Bacteriófago P22/química , Bacteriófago P22/ultraestructura , Proteínas de la Cápside/química , Proteínas de la Cápside/ultraestructura , Microscopía por Crioelectrón , Modelos Moleculares , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína , Electricidad EstáticaRESUMEN
Single particle analysis is a valuable tool in cryo-electron microscopy for determining the structure of biological complexes. However, the conformational state and the preparation of the sample are factors that play a critical role in the ultimate attainable resolution. In some cases extensive analysis at the microscope of a sample under different conditions is required to derive the optimal acquisition conditions. Currently this analysis is limited to raw micrographs, thus conveying only limited information on the structure of the complex. We are developing a computing system that generates a three-dimensional reconstruction from a single micrograph acquired under cryogenic and low dose conditions, and containing particles with icosahedral symmetry. The system provides the microscopist with immediate structural information from a sample while it is in the microscope and during the preliminary acquisition stage. The system is designed to run without user intervention on a multi-processor computing resource and integrates all the processing steps required for the analysis. Tests performed on experimental data sets show that the probability of obtaining a reliable reconstruction from one micrograph is primarily determined by the quality of the sample, with success rates close to 100% when sample conditions are optimal, and decreasing to about 60% when conditions are sub-optimal. The time required to generate a reconstruction depends significantly on the diameter of the particles, and in most instances takes about 1min. The proposed approach can provide valuable three-dimensional information, albeit at low resolution, on conformational states, epitope binding, and stoichiometry of icosahedral multi-protein complexes.
Asunto(s)
Imagenología Tridimensional , Programas Informáticos , Bacteriófago P22/ultraestructura , Cápside/ultraestructura , Microscopía por Crioelectrón/métodos , Modelos Moleculares , Estructura Cuaternaria de Proteína , Reproducibilidad de los ResultadosRESUMEN
The hierarchical organization of inorganic nanostructures has potential applications in diverse areas such as photocatalytic systems, composites, drug delivery and biomedicine. An attractive approach for this purpose is the use of biological organisms as templates since they often possess highly ordered arrays of protein molecules that can be genetically engineered for specific binding. Indeed, recent studies have shown that viruses can be used as versatile templates for the assembly of a variety of nanostructured materials because of their unique structural and chemical diversity. These highly ordered protein templates can be employed or adapted for specific binding interactions. Herein we report the directed self-assembly of independently synthesized 5 nm CdS nanocrystal quantum dots on â¼60 nm procapsid shells derived from wild-type P22 bacteriophage. The bacteriophage P22 shell is comprised of hexameric and pentameric clusters of subunits known as capsomeres. The pre-synthesized CdS QDs show the corresponding hexameric and pentameric patterns of assembly on these P22 shells, possibly by interacting with particular protein pockets.
Asunto(s)
Bacteriófago P22/química , Bacteriófago P22/ultraestructura , Compuestos de Cadmio/química , Proteínas de la Cápside/química , Proteínas de la Cápside/ultraestructura , Nanoestructuras/química , Puntos Cuánticos , Compuestos de Selenio/química , Adsorción , Cristalización/métodos , Ensayo de Materiales , Impresión Molecular/métodos , Nanoestructuras/ultraestructura , Tamaño de la PartículaRESUMEN
Assembly of icosahedral capsids of proper size and symmetry is not understood. Residue F170 in bacteriophage P22 coat protein is critical for conformational switching during assembly. Substitutions at this site cause assembly of tubes of hexamerically arranged coat protein. Intragenic suppressors of the ts phenotype of F170A and F170K coat protein mutants were isolated. Suppressors were repeatedly found in the coat protein telokin-like domain at position 285, which caused coat protein to assemble into petite procapsids and capsids. Petite capsid assembly strongly correlated to the side chain volume of the substituted amino acid. We hypothesize that larger side chains at position 285 torque the telokin-like domain, changing flexibility of the subunit and intercapsomer contacts. Thus, a single amino acid substitution in coat protein is sufficient to change capsid size. In addition, the products of assembly of the variant coat proteins were affected by the size of the internal scaffolding protein.
Asunto(s)
Bacteriófago P22/fisiología , Proteínas de la Cápside/metabolismo , Cápside/fisiología , Proteínas Estructurales Virales/metabolismo , Ensamble de Virus , Sustitución de Aminoácidos/genética , Bacteriófago P22/metabolismo , Bacteriófago P22/ultraestructura , Cápside/metabolismo , Cápside/ultraestructura , Proteínas de la Cápside/genética , Microscopía Electrónica , Modelos Moleculares , Mutación Missense , Quinasa de Cadena Ligera de Miosina/genética , Fragmentos de Péptidos/genética , Estructura Terciaria de Proteína , Supresión GenéticaRESUMEN
DNA viruses such as bacteriophages and herpesviruses deliver their genome into and out of the capsid through large proteinaceous assemblies, known as portal proteins. Here, we report two snapshots of the dodecameric portal protein of bacteriophage P22. The 3.25-Å-resolution structure of the portal-protein core bound to 12 copies of gene product 4 (gp4) reveals a ~1.1-MDa assembly formed by 24 proteins. Unexpectedly, a lower-resolution structure of the full-length portal protein unveils the unique topology of the C-terminal domain, which forms a ~200-Å-long α-helical barrel. This domain inserts deeply into the virion and is highly conserved in the Podoviridae family. We propose that the barrel domain facilitates genome spooling onto the interior surface of the capsid during genome packaging and, in analogy to a rifle barrel, increases the accuracy of genome ejection into the host cell.
Asunto(s)
Bacteriófago P22/ultraestructura , Proteínas de la Cápside/química , Secuencia de Aminoácidos , Bacteriófago P22/genética , Bacteriófago P22/metabolismo , Proteínas de la Cápside/ultraestructura , Secuencia Conservada , Cristalografía por Rayos X , ADN Viral/química , Modelos Moleculares , Pliegue de Proteína , Estructura Terciaria de Proteína , Virión/metabolismo , Virión/ultraestructuraRESUMEN
The encapsidated genome in all double-strand DNA bacteriophages is packaged to liquid crystalline density through a unique vertex in the procapsid assembly intermediate, which has a portal protein dodecamer in place of five coat protein subunits. The portal orchestrates DNA packaging and exit, through a series of varying interactions with the scaffolding, terminase, and closure proteins. Here, we report an asymmetric cryoEM reconstruction of the entire P22 virion at 7.8 Å resolution. X-ray crystal structure models of the full-length portal and of the portal lacking 123 residues at the C terminus in complex with gene product 4 (Δ123portal-gp4) obtained by Olia et al. (2011) were fitted into this reconstruction. The interpreted density map revealed that the 150 Å, coiled-coil, barrel portion of the portal entraps the last DNA to be packaged and suggests a mechanism for head-full DNA signaling and transient stabilization of the genome during addition of closure proteins.
Asunto(s)
Bacteriófago P22/química , Proteínas de la Cápside/química , Conformación Proteica , Virión/química , Bacteriófago P22/genética , Bacteriófago P22/ultraestructura , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN Viral/química , ADN Viral/genética , Genoma Viral/genética , Modelos Moleculares , Mutación , Unión Proteica , Proteínas Virales/química , Proteínas Virales/metabolismo , Virión/genética , Virión/ultraestructura , Ensamble de Virus/genéticaRESUMEN
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 = 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.
Asunto(s)
Proteínas de la Cápside/química , Virus ADN/metabolismo , Conformación Proteica , Ensamble de Virus , Bacteriófago P22/genética , Bacteriófago P22/metabolismo , Bacteriófago P22/ultraestructura , Sitios de Unión , Cápside/química , Cápside/metabolismo , Cápside/ultraestructura , Proteínas de la Cápside/metabolismo , Microscopía por Crioelectrón , Virus ADN/genética , Virus ADN/ultraestructura , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Proteínas del Núcleo Viral , Proteínas Virales/química , Proteínas Virales/metabolismo , Virión/genética , Virión/metabolismo , Virión/ultraestructuraRESUMEN
Bacteriophage P22 forms an isometric capsid during normal assembly, yet when the coat protein (CP) is altered at a single site, helical structures (polyheads) also form. The structures of three distinct polyheads obtained from F170L and F170A variants were determined by cryo-reconstruction methods. An understanding of the structures of aberrant assemblies such as polyheads helps to explain how amino acid substitutions affect the CP, and these results can now be put into the context of CP pseudo-atomic models. F170L CP forms two types of polyhead and each has the CP organized as hexons (oligomers of six CPs). These hexons have a skewed structure similar to that in procapsids (precursor capsids formed prior to dsDNA packaging), yet their organization differs completely in polyheads and procapsids. F170A CP forms only one type of polyhead, and though this has hexons organized similarly to hexons in F170L polyheads, the hexons are isometric structures like those found in mature virions. The hexon organization in all three polyheads suggests that nucleation of procapsid assembly occurs via a trimer of CP monomers, and this drives formation of a T = 7, isometric particle. These variants also form procapsids, but they mature quite differently: F170A expands spontaneously at room temperature, whereas F170L requires more energy. The P22 CP structure along with scaffolding protein interactions appear to dictate curvature and geometry in assembled structures and residue 170 significantly influences both assembly and maturation.
Asunto(s)
Bacteriófago P22/fisiología , Proteínas de la Cápside/metabolismo , Ensamble de Virus , Bacteriófago P22/metabolismo , Bacteriófago P22/ultraestructura , Cápside , Proteínas de la Cápside/química , Microscopía Electrónica de Transmisión , Modelos Moleculares , Unión ProteicaRESUMEN
The use of sub-nanometer resolution electron density as spatial constraints for de novo and ab initio structure prediction requires knowledge of protein boundaries to accurately segment the electron density for the prediction algorithms. Here we present a procedure where even poorly segmented density can be used to determine the fold of the protein. The method is automated, fast, capable of searching for multiple copies of a protein fold, and accessible to densities encompassing more than a thousand residues. The automation is particularly powerful as it allows the procedure to take full advantage of the expanding repository in the Protein Data Bank. We have tested the method on nine segmented sub-nanometer image reconstruction electron densities. The method successfully identifies the correct fold for the six densities for which an atomic structure is known, identifies a fold that agrees with prior structural data, a fold that agrees with predictions from the Fold & Function Assignment server, and a fold that correlates with secondary structure prediction. The identified folds in the last three examples can be used as templates for comparative modeling of the bacteriophage P22 tail-machine (a 3MDa complex composed of 39 protein subunits).
Asunto(s)
Pliegue de Proteína , Proteínas/química , Proteínas/ultraestructura , Algoritmos , Animales , Automatización , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Bacteriófago P22/química , Bacteriófago P22/ultraestructura , Bacteriófago lambda/química , Bacteriófago lambda/ultraestructura , Bovinos , Chaperonina 60/química , Chaperonina 60/ultraestructura , Microscopía por Crioelectrón , Cristalografía por Rayos X , Bases de Datos de Proteínas , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Modelos Moleculares , Reoviridae/química , Reoviridae/ultraestructura , Rodopsina/química , Rodopsina/ultraestructura , Diseño de Software , Electricidad EstáticaRESUMEN
The Lilleengen scheme for typing Salmonella enterica serovar Typhimurium consists of 12 tailed phages. Ten phages are podoviruses and morphologically identical to Salmonella phage P22. Two phages are siphoviruses and identical to flagella-specific phage chi.