RESUMEN

Most of studied bacteriophages (phages) are terrestrial viruses. However, marine phages are shown to be highly involved in all levels of oceanic regulation. They are, however, still largely overlooked by the scientific community. By inducing cell lysis on half of the bacterial population daily, their role and influence on the bacterial biomass and evolution, as well as their impact in the global biogeochemical cycles, is undeniable. Cobetia marina virus 1 (Carin-1) is a member of the Podoviridae family infecting the γ-protoabacteria C. marina. Here, we present the almost complete, nearly-atomic resolution structure of Carin-1 comprising capsid, portal, and tail machineries at 3.5 Å, 3.8 Å and 3.9 Å, respectively, determined by cryo-electron microscopy (cryo-EM). Our experimental results, combined with AlphaFold2 (AF), allowed us to obtain the nearly-atomic structure of Carin-1 by fitting and refining the AF atomic models in the high resolution cryo-EM map, skipping the bottleneck of de-novo manual building and speeding up the structure determination process. Our structural results highlighted the T7-like nature of Carin1, as well as several novel structural features like the presence of short spikes on the capsid, reminiscent those described for Rhodobacter capsulatus gene transfer agent (RcGTA). This is, to our knowledge, the first time such assembly is described for a bacteriophage, shedding light into the common evolution and shared mechanisms between gene transfer agents and phages. This first full structure determined for a marine podophage allowed to propose an infection mechanism different than the one proposed for the archetypal podophage T7. IMPORTANCE Oceans play a central role in the carbon cycle on Earth and on the climate regulation (half of the planet's CO2 is absorbed by phytoplankton photosynthesis in the oceans and just as much O2 is liberated). The understanding of the biochemical equilibriums of marine biology represents a major goal for our future. By lysing half of the bacterial population every day, marine bacteriophages are key actors of these equilibriums. Despite their importance, these marine phages have, so far, only been studied a little and, in particular, structural insights are currently lacking, even though they are fundamental for the understanding of the molecular mechanisms of their mode of infection. The structures described in our manuscript allow us to propose an infection mechanism that differs from the one proposed for the terrestrial T7 virus, and might also allow us to, in the future, better understand the way bacteriophages shape the global ecosystem.

Asunto(s)

Bacteriófagos , Podoviridae , Bacteriófagos/clasificación , Bacteriófagos/ultraestructura , Microscopía por Crioelectrón , Podoviridae/ultraestructura , Cápside/ultraestructura , Proteínas de la Cola de los Virus/ultraestructura , Halomonadaceae/virología

RESUMEN

Phage G is recognized as having a remarkably large genome and capsid size among isolated, propagated phages. Negative stain electron microscopy of the host-phage G interaction reveals tail sheaths that are contracted towards the distal tip and decoupled from the head-neck region. This is different from the typical myophage tail contraction, where the sheath contracts upward, while being linked to the head-neck region. Our cryo-EM structures of the non-contracted and contracted tail sheath show that: (1) The protein fold of the sheath protein is very similar to its counterpart in smaller, contractile phages such as T4 and phi812; (2) Phage G's sheath structure in the non-contracted and contracted states are similar to phage T4's sheath structure. Similarity to other myophages is confirmed by a comparison-based study of the tail sheath's helical symmetry, the sheath protein's evolutionary timetree, and the organization of genes involved in tail morphogenesis. Atypical phase G tail contraction could be due to a missing anchor point at the upper end of the tail sheath that allows the decoupling of the sheath from the head-neck region. Explaining the atypical tail contraction requires further investigation of the phage G sheath anchor points.

Asunto(s)

Myoviridae/ultraestructura , Proteínas de la Cola de los Virus/ultraestructura , Bacteriófagos/metabolismo , Bacteriófagos/ultraestructura , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Microscopía por Crioelectrón/métodos , Myoviridae/genética , Proteínas de la Cola de los Virus/genética , Proteínas de la Cola de los Virus/metabolismo , Virión/metabolismo , Virión/ultraestructura

RESUMEN

The genome of Escherichia coli O157:H7 bacteriophage vB_EcoM_CBA120 encodes four distinct tailspike proteins (TSPs). The four TSPs, TSP1-4, attach to the phage baseplate forming a branched structure. We report the 1.9 Å resolution crystal structure of TSP2 (ORF211), the TSP that confers phage specificity towards E. coli O157:H7. The structure shows that the N-terminal 168 residues involved in TSPs complex assembly are disordered in the absence of partner proteins. The ensuing head domain contains only the first of two fold modules seen in other phage vB_EcoM_CBA120 TSPs. The catalytic site resides in a cleft at the interface between adjacent trimer subunits, where Asp506, Glu568, and Asp571 are located in close proximity. Replacement of Asp506 and Asp571 for alanine residues abolishes enzyme activity, thus identifying the acid/base catalytic machinery. However, activity remains intact when Asp506 and Asp571 are mutated into asparagine residues. Analysis of additional site-directed mutants in the background of the D506N:D571N mutant suggests engagement of an alternative catalytic apparatus comprising Glu568 and Tyr623. Finally, we demonstrate the catalytic role of two interacting glutamate residues of TSP1, located in a cleft between two trimer subunits, Glu456 and Glu483, underscoring the diversity of the catalytic apparatus employed by phage vB_EcoM_CBA120 TSPs.

Asunto(s)

Bacteriófagos/genética , Escherichia coli O157/genética , Proteínas de la Cola de los Virus/ultraestructura , Bacteriófagos/metabolismo , Bacteriófagos/patogenicidad , Dominio Catalítico , Escherichia coli O157/metabolismo , Glicósido Hidrolasas , Especificidad de la Especie , Proteínas de la Cola de los Virus/genética , Proteínas de la Cola de los Virus/metabolismo , Virión

RESUMEN

Virulent phages infecting L. lactis, an industry-relevant bacterium, pose a significant risk to the quality of the fermented milk products. Phages of the Skunavirus genus are by far the most isolated lactococcal phages in the cheese environments and phage p2 is the model siphophage for this viral genus. The baseplate of phage p2, which is used to recognize its host, was previously shown to display two conformations by X-ray crystallography, a rested state and an activated state ready to bind to the host. The baseplate became only activated and opened in the presence of Ca2+. However, such an activated state was not previously observed in the virion. Here, using nanobodies binding to the baseplate, we report on the negative staining electron microscopy structure of the activated form of the baseplate directly observed in the p2 virion, that is compatible with the activated baseplate crystal structure. Analyses of this new structure also established the presence of a second distal tail (Dit) hexamer as a component of the baseplate, the topology of which differs largely from the first one. We also observed an uncoupling between the baseplate activation and the tail tip protein (Tal) opening, suggesting an infection mechanism more complex than previously expected.

Asunto(s)

Bacteriófagos/química , Lactococcus lactis/virología , Proteínas de la Cola de los Virus/química , Bacteriófagos/genética , Cristalografía por Rayos X , Microscopía Electrónica , Modelos Moleculares , Unión Proteica , Conformación Proteica , Anticuerpos de Dominio Único/química , Proteínas de la Cola de los Virus/ultraestructura

RESUMEN

The long flexible tail tube of bacteriophage lambda connects its capsid to the tail tip. On infection, a DNA ejection signal is passed from the tip, along the tube to the capsid that triggers passage of the DNA down the tube and into the host bacterium. The tail tube is built from repeating units of the major tail protein, gpV, which has two distinctive domains. Its N-terminal domain has the same fold as proteins that form the rigid inner tubes of contractile tail phages, such as T4, and its C-terminal domain adopt an Ig-like fold of unknown function. We determined structures of the lambda tail tube in free tails and in virions before and after DNA ejection using cryoelectron microscopy. Modeling of the density maps reveals how electrostatic interactions and a mobile loop participate in assembly and also impart flexibility to the tube while maintaining its integrity. We also demonstrate how a common protein fold produces rigid tubes in some phages but flexible tubes in others.

Asunto(s)

Bacteriófago lambda/ultraestructura , Proteínas de la Cápside/ultraestructura , Siphoviridae/ultraestructura , Proteínas de la Cola de los Virus/ultraestructura , Secuencia de Aminoácidos/genética , Bacteriófago lambda/genética , Cápside/química , Cápside/ultraestructura , Proteínas de la Cápside/genética , Microscopía por Crioelectrón , Modelos Moleculares , Siphoviridae/genética , Electricidad Estática , Proteínas de la Cola de los Virus/genética , Virión/genética , Virión/ultraestructura

RESUMEN

The vast majority of phages, bacterial viruses, possess a tail ensuring host recognition, cell wall perforation and safe viral DNA transfer from the capsid to the host cytoplasm. Long flexible tails are formed from the tail tube protein (TTP) polymerised as hexameric rings around and stacked along the tape measure protein (TMP). Here, we report the crystal structure of T5 TTP pb6 at 2.2 Å resolution. Pb6 is unusual in forming a trimeric ring, although structure analysis reveals homology with all classical TTPs and related tube proteins of bacterial puncturing devices (type VI secretion system and R-pyocin). Structures of T5 tail tubes before and after interaction with the host receptor were determined by cryo-electron microscopy at 6 Å resolution. Comparison of these two structures reveals that host-binding information is not propagated to the capsid through conformational changes in the tail tube, suggesting a role of the TMP in this information transduction process.

Asunto(s)

Bacteriófagos/ultraestructura , ADN Viral/metabolismo , Siphoviridae/ultraestructura , Proteínas de la Cola de los Virus/ultraestructura , Cápside/metabolismo , Microscopía por Crioelectrón , Citoplasma/metabolismo , Escherichia coli , Homología Estructural de Proteína

RESUMEN

Bacteriophage replication requires specific host-recognition. Some siphophages harbour a large complex, the baseplate, at the tip of their non-contractile tail. This baseplate holds receptor binding proteins (RBPs) that can recognize the host cell-wall polysaccharide (CWPS) and specifically attach the phage to its host. While most phages possess a dedicated RBP, the phage J-1 that infects Lactobacillus casei seemed to lack one. It has been shown that the phage J-1 distal tail protein (Dit) plays a role in host recognition and that its sequence comprises two inserted modules compared with 'classical' Dits. The first insertion is similar to carbohydrate-binding modules (CBMs), whereas the second insertion remains undocumented. Here, we determined the structure of the second insertion and found it also similar to several CBMs. Expressed insertion CBM2, but not CBM1, binds to L. casei cells and neutralize phage attachment to the bacterial cell wall and the isolated and purified CWPS of L. casei BL23 prevents CBM2 attachment to the host. Electron microscopy single particle reconstruction of the J-1 virion baseplate revealed that CBM2 is projected at the periphery of Dit to optimally bind the CWPS receptor. Taken together, these results identify J-1 evolved Dit as the phage RBP.

Asunto(s)

Proteínas de la Cola de los Virus/metabolismo , Proteínas de la Cola de los Virus/ultraestructura , Bacteriófagos/metabolismo , Carbohidratos , Especificidad del Huésped , Ácido Láctico , Lactobacillus , Lacticaseibacillus casei/metabolismo , Lactococcus lactis/metabolismo , Microscopía Electrónica , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Proteínas de la Cola de los Virus/genética , Virión

RESUMEN

Most bacteriophages are tailed bacteriophages with an isometric or a prolate head attached to a long contractile, long non-contractile, or short non-contractile tail. The tail is a complex machine that plays a central role in host cell recognition and attachment, cell wall and membrane penetration, and viral genome ejection. The mechanisms involved in the penetration of the inner host cell membrane by bacteriophage tails are not well understood. Here we describe structural and functional studies of the bacteriophage ϕ29 tail knob protein gene product 9 (gp9). The 2.0 Šcrystal structure of gp9 shows that six gp9 molecules form a hexameric tube structure with six flexible hydrophobic loops blocking one end of the tube before DNA ejection. Sequence and structural analyses suggest that the loops in the tube could be membrane active. Further biochemical assays and electron microscopy structural analyses show that the six hydrophobic loops in the tube exit upon DNA ejection and form a channel that spans the lipid bilayer of the membrane and allows the release of the bacteriophage genomic DNA, suggesting that cell membrane penetration involves a pore-forming mechanism similar to that of certain non-enveloped eukaryotic viruses. A search of other phage tail proteins identified similar hydrophobic loops, which indicates that a common mechanism might be used for membrane penetration by prokaryotic viruses. These findings suggest that although prokaryotic and eukaryotic viruses use apparently very different mechanisms for infection, they have evolved similar mechanisms for breaching the cell membrane.

Asunto(s)

Fagos de Bacillus/química , Fagos de Bacillus/metabolismo , Membrana Celular/metabolismo , Proteínas de la Cola de los Virus/química , Proteínas de la Cola de los Virus/metabolismo , Secuencia de Aminoácidos , Fagos de Bacillus/genética , Fagos de Bacillus/ultraestructura , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN Viral/metabolismo , Genoma Viral/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/química , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Porosidad , Estructura Cuaternaria de Proteína , Proteínas de la Cola de los Virus/ultraestructura , Virión/genética , Virión/ultraestructura

RESUMEN

Bacterial viruses of the P22-like family encode a specialized tail needle essential for genome stabilization after DNA packaging and implicated in Gram-negative cell envelope penetration. The atomic structure of P22 tail needle (gp26) crystallized at acidic pH reveals a slender fiber containing an N-terminal "trimer of hairpins" tip. Although the length and composition of tail needles vary significantly in Podoviridae, unexpectedly, the amino acid sequence of the N-terminal tip is exceptionally conserved in more than 200 genomes of P22-like phages and prophages. In this paper, we used x-ray crystallography and EM to investigate the neutral pH structure of three tail needles from bacteriophage P22, HK620, and Sf6. In all cases, we found that the N-terminal tip is poorly structured, in stark contrast to the compact trimer of hairpins seen in gp26 crystallized at acidic pH. Hydrogen-deuterium exchange mass spectrometry, limited proteolysis, circular dichroism spectroscopy, and gel filtration chromatography revealed that the N-terminal tip is highly dynamic in solution and unlikely to adopt a stable trimeric conformation at physiological pH. This is supported by the cryo-EM reconstruction of P22 mature virion tail, where the density of gp26 N-terminal tip is incompatible with a trimer of hairpins. We propose the tail needle N-terminal tip exists in two conformations: a pre-ejection extended conformation, which seals the portal vertex after genome packaging, and a postejection trimer of hairpins, which forms upon its release from the virion. The conformational plasticity of the tail needle N-terminal tip is built in the amino acid sequence, explaining its extraordinary conservation in nature.

Asunto(s)

Genoma Viral , Podoviridae/genética , Proteínas de la Cola de los Virus/química , Virión/genética , Ensamble de Virus , Bacteriófagos/química , Dicroismo Circular , Microscopía por Crioelectrón , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Concentración de Iones de Hidrógeno , Espectrometría de Masas , Coloración Negativa , Multimerización de Proteína , Proteínas de la Cola de los Virus/ultraestructura

RESUMEN

Each phage infects its specific bacterial host strain through highly specific interactions between the baseplate-associated receptor binding protein (RBP) at the tip of the phage tail and the receptor at the host surface. Baseplates incorporate structural core modules, Dit and Tal, largely conserved among phages, and peripheral modules anchoring the RBPs. Exploiting structural information from the HHpred program and EM data from the Bielmann et al. (2015) paper, a molecular model of the A118 phage baseplate was generated from different building blocks. This model implies the occurrence of baseplate module reshuffling and suggests that listerial phage A118 may have been derived from lactococcal phage TP901-1 through host species exchange. With the increase of available viral module structures, modelling phage baseplates will become easier and more reliant, and will provide insightful information on the nature of the phage host receptor and its mode of recognition.

Asunto(s)

Bacteriófagos/fisiología , Bacteriófagos/ultraestructura , Especificidad del Huésped , Proteínas de la Cola de los Virus/metabolismo , Proteínas de la Cola de los Virus/ultraestructura , Acoplamiento Viral , Bacteriófagos/química , Procesamiento de Imagen Asistido por Computador , Listeria monocytogenes/virología , Microscopía Electrónica , Modelos Moleculares , Conformación Proteica , Proteínas de la Cola de los Virus/química

RESUMEN

Many icosahedral viruses use a specialized portal vertex to control genome encapsidation and release from the viral capsid. In tailed bacteriophages, the portal system is connected to a tail structure that provides the pipeline for genome delivery to the host cell. We report the first, to our knowledge, subnanometer structures of the complete portal-phage tail interface that mimic the states before and after DNA release during phage infection. They uncover structural rearrangements associated with intimate protein-DNA interactions. The portal protein gp6 of bacteriophage SPP1 undergoes a concerted reorganization of the structural elements of its central channel during interaction with DNA. A network of protein-protein interactions primes consecutive binding of proteins gp15 and gp16 to extend and close the channel. This critical step that prevents genome leakage from the capsid is achieved by a previously unidentified allosteric mechanism: gp16 binding to two different regions of gp15 drives correct positioning and folding of an inner gp16 loop to interact with equivalent loops of the other gp16 subunits. Together, these loops build a plug that closes the channel. Gp16 then fastens the tail to yield the infectious virion. The gatekeeper system opens for viral genome exit at the beginning of infection but recloses afterward, suggesting a molecular diaphragm-like mechanism to control DNA efflux. The mechanisms described here, controlling the essential steps of phage genome movements during virus assembly and infection, are likely to be conserved among long-tailed phages, the largest group of viruses in the Biosphere.

Asunto(s)

Bacteriófagos/química , Genoma Viral/fisiología , Modelos Moleculares , Proteínas Virales/química , Proteínas de la Cola de los Virus/química , Ensamble de Virus/fisiología , Internalización del Virus , Bacteriófagos/ultraestructura , Microscopía por Crioelectrón , Genoma Viral/genética , Conformación Proteica , Proteínas Virales/metabolismo , Proteínas Virales/ultraestructura , Proteínas de la Cola de los Virus/metabolismo , Proteínas de la Cola de los Virus/ultraestructura

RESUMEN

Protein fibers are widespread in nature, but only a limited number of high-resolution structures have been determined experimentally. Unlike globular proteins, fibers are usually recalcitrant to form three-dimensional crystals, preventing single-crystal X-ray diffraction analysis. In the absence of three-dimensional crystals, X-ray fiber diffraction is a powerful tool to determine the internal symmetry of a fiber, but it rarely yields atomic resolution structural information on complex protein fibers. An 85-residue-long minimal coiled-coil repeat unit (MiCRU) was previously identified in the trimeric helical core of tail needle gp26, a fibrous protein emanating from the tail apparatus of the bacteriophage P22 virion. Here, evidence is provided that an MiCRU can be inserted in frame inside the gp26 helical core to generate a rationally extended fiber (gp26-2M) which, like gp26, retains a trimeric quaternary structure in solution. The 2.7 Šresolution crystal structure of this engineered fiber, which measures ∼320 Šin length and is only 20-35 Šwide, was determined. This structure, the longest for a trimeric protein fiber to be determined to such a high resolution, reveals the architecture of 22 consecutive trimerization heptads and provides a framework to decipher the structural determinants for protein fiber assembly, stability and flexibility.

Asunto(s)

Bacteriófago P22/química , Ingeniería de Proteínas , Proteínas de la Cola de los Virus/ultraestructura , Secuencia de Aminoácidos , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Docilidad , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestructura , Proteínas de la Cola de los Virus/genética

RESUMEN

ϕRSL1 jumbo phage belongs to a new class of viruses within the Myoviridae family. Here, we report its three-dimensional structure determined by electron cryo microscopy. The icosahedral capsid, the tail helical portion, and the complete tail appendage were reconstructed separately to resolutions of 9 Å, 9 Å, and 28 Å, respectively. The head is rather complex and formed by at least five different proteins, whereas the major capsid proteins resemble those from HK97, despite low sequence conservation. The helical tail structure demonstrates its close relationship to T4 sheath proteins and provides evidence for an evolutionary link of the inner tail tube to the bacterial type VI secretion apparatus. Long fibers extend from the collar region, and their length is consistent with reaching the host cell surface upon tail contraction. Our structural analyses indicate that ϕRSL1 is an unusual member of the Myoviridae that employs conserved protein machines related to different phages and bacteria.

Asunto(s)

Bacteriófagos/ultraestructura , Ralstonia solanacearum/virología , Cápside/ultraestructura , Proteínas de la Cápside/ultraestructura , Microscopía por Crioelectrón , Modelos Moleculares , Estructura Cuaternaria de Proteína , Proteínas de la Cola de los Virus/ultraestructura

RESUMEN

The structure of the Bacillus anthracis spore-binding phage 8a was determined by cryo-electron tomography. The phage capsid forms a T=16 icosahedron attached to a contractile tail via a head-tail connector protein. The tail consists of a six-start helical sheath surrounding a central tail tube, and a structurally novel baseplate at the distal end of the tail that recognizes and attaches to host cells. The parameters of the icosahedral capsid lattice and the helical tail sheath suggest protein folds for the capsid and tail-sheath proteins, respectively, and indicate evolutionary relationships to other dsDNA viruses. Analysis of 2518 intact phage particles show four distinct conformations that likely correspond to four sequential states of the DNA ejection process during infection. Comparison of the four observed conformations suggests a mechanism for DNA ejection, including the molecular basis underlying coordination of tail sheath contraction and genome release from the capsid.

Asunto(s)

Fagos de Bacillus/fisiología , Fagos de Bacillus/ultraestructura , ADN Viral/metabolismo , Myoviridae/fisiología , Myoviridae/ultraestructura , Fagos de Bacillus/química , Fagos de Bacillus/genética , Bacillus anthracis/virología , Cápside/ultraestructura , Proteínas de la Cápside/química , Proteínas de la Cápside/ultraestructura , Tomografía con Microscopio Electrónico , Myoviridae/química , Myoviridae/genética , Esporas Bacterianas/virología , Proteínas de la Cola de los Virus/química , Proteínas de la Cola de los Virus/ultraestructura

RESUMEN

Vibriophage D10, a member of the Vibrio cholerae O-1El-Tor phage typing scheme, is used to detect the spread of the cholera epidemic and belongs to the Myoviridae family. The outer sheath of the tail of vibriophage is highly contractile in nature. We have used electron microscopy and computational image-processing techniques to determine the structure of this contractile tail sheath. The three-dimensional density map of the tail sheath reveals the presence of ∼35 Å long and ∼25 Å wide protrusions, extending out of the tail structure. The electron micrographs revealed that the tail sheaths of a considerable number of D10 phage particles undergo axial compression up to 51% at almost neutral pH (7.2) and at room temperature (20°). We find that the genome of the phage particles is ejected out when the tail sheath of the phage particles are deliberately made to contract by subjecting them to a surrounding environment of pH 10.5. We infer that the contraction of the tail sheath is responsible for the loss of the phage genome even at neutral pH and room temperature. This may be a plausible reason for the unusual behavior of rapid decline of the phage within a span of 48-72 h of its preparation.

Asunto(s)

Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica , Myoviridae/ultraestructura , Vibrio cholerae/virología , Proteínas de la Cola de los Virus/ultraestructura , Genoma Viral , Concentración de Iones de Hidrógeno , Myoviridae/genética

RESUMEN

Remarkable progress has been made during the past ten years in elucidating the structure of the bacteriophage T4 tail by a combination of three-dimensional image reconstruction from electron micrographs and X-ray crystallography of the components. Partial and complete structures of nine out of twenty tail structural proteins have been determined by X-ray crystallography and have been fitted into the 3D-reconstituted structure of the "extended" tail. The 3D structure of the "contracted" tail was also determined and interpreted in terms of component proteins. Given the pseudo-atomic tail structures both before and after contraction, it is now possible to understand the gross conformational change of the baseplate in terms of the change in the relative positions of the subunit proteins. These studies have explained how the conformational change of the baseplate and contraction of the tail are related to the tail's host cell recognition and membrane penetration function. On the other hand, the baseplate assembly process has been recently reexamined in detail in a precise system involving recombinant proteins (unlike the earlier studies with phage mutants). These experiments showed that the sequential association of the subunits of the baseplate wedge is based on the induced-fit upon association of each subunit. It was also found that, upon association of gp53 (gene product 53), the penultimate subunit of the wedge, six of the wedge intermediates spontaneously associate to form a baseplate-like structure in the absence of the central hub. Structure determination of the rest of the subunits and intermediate complexes and the assembly of the hub still require further study.

Asunto(s)

Bacteriófago T4/química , Bacteriófago T4/ultraestructura , Sustancias Macromoleculares/química , Sustancias Macromoleculares/ultraestructura , Proteínas de la Cola de los Virus/química , Proteínas de la Cola de los Virus/ultraestructura , Cristalografía por Rayos X , Imagenología Tridimensional , Microscopía Electrónica , Modelos Biológicos , Modelos Moleculares , Myoviridae/química , Myoviridae/ultraestructura

RESUMEN

The portal channel is a key component in the life cycle of bacteriophages and herpesviruses. The bacteriophage P22 portal is a 1 megadalton dodecameric oligomer of gp1 that plays key roles in capsid assembly, DNA packaging, assembly of the infection machinery, and DNA ejection. The portal is the nucleation site for the assembly of 39 additional subunits generated from multiple copies of four gene products (gp4, gp10, gp9, and gp26), which together form the multifunctional tail machine. These components are organized with a combination of 12-fold (gp1, gp4), 6-fold (gp10, trimers of gp9), and 3-fold (gp26, gp9) symmetry. Here we present the 3-dimensional structures of the P22 assembly-naive portal formed from expressed subunits (gp1) and the intact tail machine purified from infectious virions. The assembly-naive portal structure exhibits a striking structural similarity to the structures of the portal proteins of SPP1 and phi29 derived from X-ray crystallography.

Asunto(s)

Bacteriófago P22/química , Bacteriófago P22/metabolismo , Proteínas de la Cola de los Virus/química , Proteínas de la Cola de los Virus/metabolismo , Virión , Bacteriófago P22/genética , Bacteriófago P22/ultraestructura , Sitios de Unión , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas de la Cola de los Virus/genética , Proteínas de la Cola de los Virus/ultraestructura

RESUMEN

Two helical samples: F-actin and the bacteriophage T4 tail sheath were reconstructed in three dimensions from contrast enhanced (rotational shadowing and negatively stained) in-lens cryo-field emission scanning electron micrographs, using the iterative real-space helical reconstruction method. The F-actin--and bacteriophage T4 reconstructions compare favourably to an atomic model refined against fibre diffraction data and a cryo-electron microscopy reconstruction, respectively. These results show that single-particle methods, developed for macromolecules imaged in the transmission electron microscope can be applied to cryo-field emission scanning electron micrographs data with appropriate symmetry.

Asunto(s)

Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Sustancias Macromoleculares/química , Microscopía Electrónica de Rastreo/métodos , Actinas/química , Actinas/ultraestructura , Bacteriófago T4/química , Bacteriófago T4/ultraestructura , Estructura Cuaternaria de Proteína , Proteínas de la Cola de los Virus/química , Proteínas de la Cola de los Virus/ultraestructura

RESUMEN

A common objective in protein engineering is the enhancement of the thermodynamic properties of recombinant proteins for possible applications in nanobiotechnology. The performance of proteins can be improved by the rational design of chimeras that contain structural elements with the desired properties, thus resulting in a more effective exploitation of protein folds designed by nature. In this paper, we report the design and characterization of an ultra-stable self-refolding protein fiber, which rapidly reassembles in solution after denaturation induced by harsh chemical treatment or high temperature. This engineered protein fiber was constructed on the molecular framework of bacteriophage P22 tail needle gp26, by fusing its helical core to the foldon domain of phage T4 fibritin. Using protein engineering, we rationally permuted the foldon upstream and downstream from the gp26 helical core and characterized gp26-foldon chimeras by biophysical analysis. Our data demonstrate that one specific protein chimera containing the foldon immediately downstream from the gp26 helical core, gp26(1-140)-F, displays the highest thermodynamic and structural stability and refolds spontaneously in solution following denaturation. The gp26-foldon chimeric fiber remains stable in 6.0 M guanidine hydrochloride, or at 80 degrees C, rapidly refolds after denaturation, and has both N and C termini accessible for chemical/biological modification, thereby representing an ideal platform for the design of self-assembling nanoblocks.

Asunto(s)

Bacteriófago T4/química , Pliegue de Proteína , Proteínas/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Bacteriófago P22/química , Bacteriófago P22/metabolismo , Bacteriófago T4/metabolismo , Escherichia coli/genética , Guanidina/metabolismo , Modelos Químicos , Desnaturalización Proteica , Ingeniería de Proteínas/métodos , Renaturación de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/aislamiento & purificación , Proteínas/ultraestructura , Temperatura , Termodinámica , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificación , Proteínas Virales/ultraestructura , Proteínas de la Cola de los Virus/química , Proteínas de la Cola de los Virus/genética , Proteínas de la Cola de los Virus/aislamiento & purificación , Proteínas de la Cola de los Virus/metabolismo , Proteínas de la Cola de los Virus/ultraestructura

RESUMEN

Structural comparisons between bacteriophage PRD1 and adenovirus have revealed an evolutionary relationship that has contributed significantly to current ideas on virus phylogeny. However, the structural organization of the receptor-binding spike complex and how the different symmetry mismatches are mediated between the spike-complex proteins are not clear. We determined the architecture of the PRD1 spike complex by using electron microscopy and three-dimensional image reconstruction of a series of PRD1 mutants. We constructed an atomic model for the full-length P5 spike protein by using comparative modeling. P5 was shown to be bound directly to the penton base protein P31. P5 and the receptor-binding protein P2 form two separate spikes, interacting with each other near the capsid shell. P5, with a tumor necrosis factor-like head domain, may have been responsible for host recognition before capture of the current receptor-binding protein P2.

Asunto(s)

Bacteriófago PRD1/química , Proteínas de la Cápside/química , Proteínas de la Cola de los Virus/química , Bacteriófago PRD1/ultraestructura , Proteínas de la Cápside/ultraestructura , Microscopía por Crioelectrón , Glicósido Hidrolasas , Modelos Moleculares , Proteínas de la Cola de los Virus/ultraestructura