The bacteriophage T4 baseplate is the control center of the virus, where the recognition of an E. coli host by the long tail fibers is translated into a signal to initiate infection. The short tail fibers unfold from the baseplate for firm attachment to the host, followed by shrinkage of the tail sheath that causes the tail tube to enter and cross the periplasmic space ending with injection of the genome into the host. During this process, the 6.5 MDa baseplate changes its structure from a “dome” shape to a “star” shape. An in vitro assembled hubless baseplate has been crystalized. It consists of six copies of the recombinantly expressed trimeric gene product (gp) 10, monomeric gp7, dimeric gp8, dimeric gp6 and monomeric gp53. The diffraction pattern extends, at most, to 4.0 Å resolution. The known partial structures of gp10, gp8, and gp6 and their relative position in the baseplate derived from earlier electron microscopy studies were used for molecular replacement. An electron density map has been calculated based on molecular replacement, single isomorphous replacement with anomalous dispersion data and 2-fold non-crystallographic symmetry averaging between two baseplate wedges in the crystallographic asymmetric unit. The current electron density map indicates that there are structural changes in the gp6, gp8, and gp10 oligomers compared to their structures when separately crystallized. Additional density is also visible corresponding to gp7, gp53 and the unknown parts of gp10 and gp6.
Bacteriophage T4; baseplate; X-ray crystallography; low resolution
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
•NADH oxidase and AhpC (Prx) form an oligomeric complex depending on ionic strength of ammonium sulfate.•The complex formation is required for NADH oxidase–Prx system to rapidly reduce hydroperoxides.•The solution structure of the complex was observed by SAXS analysis.
The NADH oxidase–peroxiredoxin (Prx) system of Amphibacillus xylanus reduces hydroperoxides with the highest turnover rate among the known hydroperoxide-scavenging enzymes. The high electron transfer rate suggests that there exists close interaction between NADH oxidase and Prx. Variant enzyme experiments indicated that the electrons from β-NADH passed through the secondary disulfide, Cys128–Cys131, of NADH oxidase to finally reduce Prx. We previously reported that ionic strength is essential for a system to reduce hydroperoxides. In this study, we analyzed the effects of ammonium sulfate (AS) on the interaction between NADH oxidase and Prx by surface plasmon resonance analysis. The interaction between NADH oxidase and Prx was observed in the presence of AS. Dynamic light scattering assays were conducted while altering the concentration of AS and the ratio of NADH oxidase to Prx in the solutions. The results revealed that the two proteins formed a large oligomeric assembly, the size of which depended on the ionic strength of AS. The molecular mass of the assembly converged at approximately 300 kDa above 240 mM AS. The observed reduction rate of hydrogen peroxide also converged at the same concentration of AS, indicating that a complex formation is required for activation of the enzyme system. That the complex generation is dependent on ionic strength was confirmed by ultracentrifugal analysis, which resulted in a signal peak derived from a complex of NADH oxidase and Prx (300 mM AS, NADH oxidase: Prx = 1:10). The complex formation under this condition was also confirmed structurally by small-angle X-ray scattering.
Nox, NADH oxidase; AhpC (Prx), peroxiredoxin; AS, ammonium sulfate; SPR, surface plasmon resonance; DLS, dynamic light scattering; AUC, analytical ultracentrifugation; SAXS, small-angle X-ray scattering; Amphibacillus xylanus; NADH oxidase; AhpC (Prx); Protein interaction; Ionic strength
A hexamer of the bacteriophage T4 tail terminator protein, gp15, attaches to the top of the phage tail stabilizing the contractile sheath and forming the interface for binding of the independently assembled head. Here we report the crystal structure of the gp15 hexamer, describe its interactions in T4 virions that have either an extended tail or a contracted tail, and discuss its structural relationship to other phage proteins. The neck of T4 virions is decorated by the “collar” and “whiskers”, made of fibritin molecules. Fibritin acts as a chaperone helping to attach the long tail fibers to the virus during the assembly process. The collar and whiskers are environment-sensing devices, regulating the retraction of the long tail fibers under unfavorable conditions, thus preventing infection. Cryo-electron microscopy analysis suggests that twelve fibritin molecules attach to the phage neck with six molecules forming the collar and six molecules forming the whiskers.
bacteriophage T4; fibritin; gpwac; T4 collar and whiskers; tail terminator protein, gp15
Atg38 provides a physical linkage between the Vps15–Vps34 and Atg14–Vps30 subcomplexes to facilitate PI3-kinase complex I formation.
Autophagy is a conserved eukaryotic process of protein and organelle self-degradation within the vacuole/lysosome. Autophagy is characterized by the formation of an autophagosome, for which Vps34-dervied phosphatidylinositol 3-phosphate (PI3P) is essential. In yeast, Vps34 forms two distinct protein complexes: complex I, which functions in autophagy, and complex II, which is involved in protein sorting to the vacuole. Here we identify and characterize Atg38 as a stably associated subunit of complex I. In atg38Δ cells, autophagic activity was significantly reduced and PI3-kinase complex I dissociated into the Vps15–Vps34 and Atg14–Vps30 subcomplexes. We find that Atg38 physically interacted with Atg14 and Vps34 via its N terminus. Further biochemical analyses revealed that Atg38 homodimerizes through its C terminus and that this homodimer formation is indispensable for the integrity of complex I. These data suggest that the homodimer of Atg38 functions as a physical linkage between the Vps15–Vps34 and Atg14–Vps30 subcomplexes to facilitate complex I formation.
In all eukaryotes, transcribed precursor tRNAs are maturated by processing and modification processes in nucleus and are transported to the cytoplasm. The cytoplasmic export protein (Cex1p) captures mature tRNAs from the nuclear export receptor (Los1p) on the cytoplasmic side of the nuclear pore complex, and it delivers them to eukaryotic elongation factor 1α. This conserved Cex1p function is essential for the quality control of mature tRNAs to ensure accurate translation. However, the structural basis of how Cex1p recognizes tRNAs and shuttles them to the translational apparatus remains unclear. Here, we solved the 2.2 Å resolution crystal structure of Saccharomyces cerevisiae Cex1p with C-terminal 197 disordered residues truncated. Cex1p adopts an elongated architecture, consisting of N-terminal kinase-like and a C-terminal α-helical HEAT repeat domains. Structure-based biochemical analyses suggested that Cex1p binds tRNAs on its inner side, using the positively charged HEAT repeat surface and the C-terminal disordered region. The N-terminal kinase-like domain acts as a scaffold to interact with the Ran-exportin (Los1p·Gsp1p) machinery. These results provide the structural basis of Los1p·Gsp1p·Cex1p·tRNA complex formation, thus clarifying the dynamic mechanism of tRNA shuttling from exportin to the translational apparatus.
The P9-1 protein of Rice black streaked dwarf virus accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in viruses in the family Reoviridae. Crystallographic analysis of P9-1 revealed structural features that allow the protein to form dimers via hydrophobic interactions. Each dimer has carboxy-terminal regions, resembling arms, that extend to neighboring dimers, thereby uniting sets of four dimers via lateral hydrophobic interactions, to yield cylindrical octamers. The importance of these regions for the formation of viroplasm-like inclusions was confirmed by the absence of such inclusions when P9-1 was expressed without its carboxy-terminal arm. The octamers are vertically elongated cylinders resembling the structures formed by NSP2 of rotavirus, even though there are no significant similarities between the respective primary and secondary structures of the two proteins. Our results suggest that an octameric structure with an internal pore might be important for the functioning of the respective proteins in the events that occur in the viroplasm, which might include viral morphogenesis.
A lytic phage, designated as ϕTMA, was isolated from a Japanese hot spring using Thermus thermophilus HB27 as an indicator strain. Electron microscopic examination showed that ϕTMA had an icosahedral head and a contractile tail. The circular double-stranded DNA sequence of ϕTMA was 151,483 bp in length, and its organization was essentially same as that of ϕYS40 except that the ϕTMA genome contained genes for a pair of transposase and resolvase, and a gene for a serine to asparagine substituted ortholog of the protein involved in the initiation of the ϕYS40 genomic DNA synthesis. The different host specificities of ϕTMA and ϕYS40 could be explained by the sequence differences in the C-terminal regions of their distal tail fiber proteins. The ΔpilA knockout strains of T. thermophilus showed simultaneous loss of sensitivity to their cognate phages, pilus structure, twitching motility and competence for natural transformation, thus suggesting that the phage infection required the intact host pili. Pulsed-field gel electrophoresis analysis of the ϕTMA and ϕYS40 genomes revealed that the length of their DNA exceeded 200 kb, indicating that the terminal redundancy is more than 30% of the closed circular form. Proteomic analysis of the ϕTMA virion using a combination of N-terminal sequencing and mass spectrometric analysis of peptide fragments suggested that the maturation of several proteins involved in the phage assembly process was mediated by a trypsin-like protease. The gene order of the phage structural proteins was also discussed.
Thermus thermophilus; myovirus; genomics; antagonistic coevolution; proteomics
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.
Heterochromatin protein 1 (HP1) is a conserved chromosomal protein with important roles in chromatin packaging and gene silencing. In fission yeast, two HP1 family proteins, Swi6 and Chp2, are involved in transcriptional silencing at heterochromatic regions, but how they function and whether they act cooperatively or differentially in heterochromatin assembly remain elusive. Here, we show that both Swi6 and Chp2 are required for the assembly of fully repressive heterochromatin, in which they play distinct, nonoverlapping roles. Swi6 is expressed abundantly and plays a dose-dependent role in forming a repressive structure through its self-association property. In contrast, Chp2, expressed at a lower level, does not show a simple dose-dependent repressive activity. However, it contributes to the recruitment of chromatin-modulating factors Clr3 and Epe1 and possesses a novel ability to bind the chromatin-enriched nuclear subfraction that is closely linked with its silencing function. Finally, we demonstrate that a proper balance between Swi6 and Chp2 is critical for heterochromatin assembly. Our findings provide novel insight into the distinct and cooperative functions of multiple HP1 family proteins in the formation of higher-order chromatin structure.
Dynein ATPases power diverse microtubule-based motilities. Each dynein motor domain comprises a ring-like head containing six AAA+ modules and N- and C-terminal regions, together with a stalk that binds microtubules. How these subdomains are arranged and generate force remains poorly understood. Here, using electron microscopy and image processing of tagged and truncated Dictyostelium cytoplasmic dynein constructs, we show that the heart of the motor is a hexameric ring of AAA+ modules, with the stalk emerging opposite the primary ATPase site (AAA1). The C-terminal region is not an integral part of the ring but spans between AAA6 and near the stalk base. The N-terminal region includes a lever-like linker whose N terminus swings by ∼17 nm during the ATPase cycle between AAA2 and the stalk base. Together with evidence of stalk tilting, which may communicate changes in microtubule binding affinity, these findings suggest a model for dynein's structure and mechanism.
KVP40 is a T4-related phage, composed of 386 open reading frames (ORFs), that has a broad host range. Here, we overexpressed, purified, and biophysically characterized two of the proteins encoded in the KVP40 genome, namely, gp5 and ORF334. Homology-based comparison between KVP40 and its better-characterized sister phage, T4, was used to estimate the two KVP40 proteins' functions. KVP40 gp5 shared significant homology with T4 gp5 in the N- and C-terminal domains. Unlike T4 gp5, KVP40 gp5 lacked the internal lysozyme domain. Like T4 gp5, KVP40 gp5 was found to form a homotrimer in solution. In stark contrast, KVP40 ORF334 shared no significant homology with any known proteins from T4-related phages. KVP40 ORF334 was found to form a heterohexamer with KVP40 gp5 in solution in a fashion nearly identical to the interaction between the T4 gp5 and gp27 proteins. Electron microscope image analysis of the KVP40 gp5-ORF334 complex indicated that it had dimensions very similar to those of the T4 gp5-gp27 structure. On the basis of our biophysical characterization, along with positional genome information, we propose that ORF334 is the ortholog of T4 gp27 and that it plays the role of a linker between gp5 and the phage baseplate.
The most extensive structural information on viruses relates to apparently icosahedral virions and is based on X-ray crystallography and on cryo-electron microscopy single-particle reconstructions. This paper concerns itself with the study of the macromolecular complexes that constitute viruses, using structural hybrid techniques.
The most extensive structural information on viruses relates to apparently icosahedral virions and is based on X-ray crystallography and on cryo-electron microscopy (cryo-EM) single-particle reconstructions. Both techniques lean heavily on imposing icosahedral symmetry, thereby obscuring any deviation from the assumed symmetry. However, tailed bacteriophages have icosahedral or prolate icosahedral heads that have one obvious unique vertex where the genome can enter for DNA packaging and exit when infecting a host cell. The presence of the tail allows cryo-EM reconstructions in which the special vertex is used to orient the head in a unique manner. Some very large dsDNA icosahedral viruses also develop special vertices thought to be required for infecting host cells. Similarly, preliminary cryo-EM data for the small ssDNA canine parvovirus complexed with receptor suggests that these viruses, previously considered to be accurately icosahedral, might have some asymmetric properties that generate one preferred receptor-binding site on the viral surface. Comparisons are made between rhinoviruses that bind receptor molecules uniformly to all 60 equivalent binding sites, canine parvovirus, which appears to have a preferred receptor-binding site, and bacteriophage T4, which gains major biological advantages on account of its unique vertex and tail organelle.
Bacteriophage T4; canine parvovirus; cryo-electron microscopy; image reconstruction; large dsDNA icosahedral viruses; special vertex
Two proteins, gp15 and gp3 (gp for gene product), are required to complete the assembly of the T4 tail. gp15 forms the connector which enables the tail to bind to the head, whereas gp3 is involved in terminating the elongation of the tail tube. In this work, genes 15 and 3 were cloned and overexpressed, and the purified gene products were studied by analytical ultracentrifugation, electron microscopy, and circular dichroism. Determination of oligomerization state by sedimentation equilibrium revealed that both gp15 and gp3 are hexamers of the respective polypeptide chains. Electron microscopy of the negatively stained P15 and P3 (P denotes the oligomeric state of the gene product) revealed that both proteins form hexameric rings, the diameter of which is close to that of the tail tube. The differential roles between gp15 and gp3 upon completion of the tail are discussed.
Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the “cell-puncturing device,” combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages—the most abundant and among the most ancient biological entities on Earth.
Adeno-associated virus capsids are composed of three proteins, VP1, VP2, and VP3. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but the functional roles of each protein are still not well understood. By analyzing a series of deletion mutants of VP2, we identified a region necessary for nuclear transfer of VP2 and found that the efficiency of nuclear localization of the capsid proteins and the efficiency of virus-like particle (VLP) formation correlated well. To confirm the importance of the nuclear localization of the capsid proteins, we fused the nuclear localization signal of simian virus 40 large T antigen to VP3 protein. We show that this fusion protein could form VLP, indicating that the VP2-specific region located on the N-terminal side of the protein is not structurally required. This finding suggests that VP3 has sufficient information for VLP formation and that VP2 is necessary only for nuclear transfer of the capsid proteins.
Tail-associated lysozyme of bacteriophage T4 (tail lysozyme), the product of gene 5 (gp 5), is an essential structural component of the hub of the phage baseplate. It is synthesized as a 63-kDa precursor, which later cleaves to form mature gp 5 with a molecular weight of 43,000. To elucidate the role of the C-terminal region of the precursor protein, gene 5 was cloned and overexpressed and the product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, analytical ultracentrifugation, and circular dichroism. It was shown that the precursor protein tends to be cleaved into two fragments during expression and that the cleavage site is close to or perhaps identical to the cleavage site in the infected cell. The two fragments, however, remained associated. The lysozyme activity of the precursor or the nicked protein is about 10% of that of mature gp 5. Both the N-terminal mature tail lysozyme and the C-terminal fragment were then isolated and characterized by far-UV circular dichroism and analytical ultracentrifugation. The latter remained trimeric after dissociation from the N-terminal fragment and is rich in β-structure as predicted by an empirical method. To trace the fate of the C-terminal fragment, antiserum was raised against a synthesized peptide of the last 12 C-terminal residues. Surprisingly, the C-terminal fragment was found in the tail and the phage particle by immunoblotting. The significance of this finding is discussed in relation to the molecular assembly and infection process.