Shuji Kanamaru

Structure of the cell-puncturing device of bacteriophage T4

Bacteriophage T4 has a very efficient mechanism for infecting cells. The key component of this process is the baseplate, located at the end of the phage tail, which regulates the interaction of the tail fibres and the DNA ejection machine. A complex of gene product (gp) 5 (63K) and gp27 (44K), the central part of the baseplate, is required to penetrate the outer cell membrane of Escherichia coli and to disrupt the intermembrane peptidoglycan layer, promoting subsequent entry of phage DNA into the host. We present here a crystal structure of the (gp5–gp27)3 321K complex, determined to 2.9 Å resolution and fitted into a cryo-electron microscopy map at 17 Å resolution of the baseplate-tail tube assembly. The carboxy-terminal domain of gp5 is a triple-stranded β-helix that forms an equilateral triangular prism, which acts as a membrane-puncturing needle. The middle lysozyme domain of gp5, situated on the periphery of the prism, serves to digest the peptidoglycan layer. The amino-terminal, antiparallel β-barrel domain of gp5 is inserted into a cylinder formed by three gp27 monomers, which may serve as a channel for DNA ejection.

Structure and morphogenesis of bacteriophage T4.

Bacteriophage T4 is one of the most complex viruses. More than 40 different proteins form the mature virion, which consists of a protein shell encapsidating a 172-kbp double-stranded genomic DNA, a ‘tail,’ and fibers, attached to the distal end of the tail. The fibers and the tail carry the host cell recognition sensors and are required for attachment of the phage to the cell surface. The tail also serves as a channel for delivery of the phage DNA from the head into the host cell cytoplasm. The tail is attached to the unique ‘portal’ vertex of the head through which the phage DNA is packaged during head assembly. Similar to other phages, and also herpes viruses, the unique vertex is occupied by a dodecameric portal protein, which is involved in DNA packaging.

Three-dimensional structure of bacteriophage T4 baseplate

The baseplate of bacteriophage T4 is a multiprotein molecular machine that controls host cell recognition, attachment, tail sheath contraction and viral DNA ejection. We report here the three-dimensional structure of the baseplate–tail tube complex determined to a resolution of 12 Å by cryoelectron microscopy. The baseplate has a six-fold symmetric, dome-like structure ∼520 Å in diameter and ∼270 Å long, assembled around a central hub. A 940 Å–long and 96 Å–diameter tail tube, coaxial with the hub, is connected to the top of the baseplate. At the center of the dome is a needle-like structure that was previously identified as a cell puncturing device. We have identified the locations of six proteins with known atomic structures, and established the position and shape of several other baseplate proteins. The baseplate structure suggests a mechanism of baseplate triggering and structural transition during the initial stages of T4 infection.

Bacteriophage φ29 scaffolding protein gp7 before and after prohead assembly

Three-dimensional structures of the double-stranded DNA bacteriophage φ29 scaffolding protein (gp7) before and after prohead assembly have been determined at resolutions of 2.2 and 2.8 Å, respectively. Both structures are dimers that resemble arrows, with a four-helix bundle composing the arrowhead and a coiled coil forming the tail. The structural resemblance of gp7 to the yeast transcription factor GCN4 suggests a DNA-binding function that was confirmed by native gel electrophoresis. DNA binding to gp7 may have a role in mediating the structural transition from prohead to mature virus and scaffold release. A cryo-EM analysis indicates that gp7 is arranged inside the capsid as a series of concentric shells. The position of the higher density features in these shells correlates with the positions of hexamers in the equatorial region of the capsid, suggesting that gp7 may regulate formation of the prolate head through interactions with these hexamers.

Zernike phase contrast cryo-electron tomography

Cryo-tomography in the electron microscope is unique in its ability to provide high-resolution, three-dimensional structural information about cells, organelles and macromolecules in a nearly native, frozen-hydrated state. However, the phase-contrast imaging method used in conventional cryo-electron tomography fails to faithfully represent the full range of structural features in such specimens. Only certain features are recorded with adequate contrast, and overall contrast is low. The recently developed Zernike phase contrast method has the potential to solve this problem, and here we apply it for the first time to cryo-electron tomography. The new method has uniform transfer characteristics for a wide range of spatial frequencies, leading to improved overall signal-to-noise ratio and raising the prospects of higher resolution and quantitative representation of specimen densities in the reconstructed tomograms.

Structural similarity of tailed phages and pathogenic bacterial secretion systems

The transport of proteins across the bacterial cell membrane is a fundamental process carried out by all groups of Gram-negative bacteria. Especially in the case of pathogenic bacteria, transport systems are used at a number of different steps in the bacterial infection pathway, such as in the export of toxins, cell adhesion, and direct penetration of effectors into the host cell. There are at least 6 distinct extracellular protein secretion systems reported as types I through VI (T1SS–T6SS) that can deliver proteins through the multilayered bacterial cell membrane and sometimes directly into the target host cell (1). The molecular organization and secretion mechanisms of type I–V secretion systems are relatively well characterized (2). However, less is known about the organization, function, and mechanism of the T6SS system first discovered by Mekalanos and coworkers in 2006 (3). After their initial report, a number of bioinformatics-based comparative analyses of the T6SS gene clusters between bacterial strains have been made to assess possible function (4–10). In this issue of PNAS, both Leiman et al. (11) and Pell et al. (12) use structure-based analysis to demonstrate the existence of a structure/function relationship between the molecular components of T6SS and the tail proteins of bacteriophages T4 (11) and λ (12).

The tail lysozyme complex of bacteriophage T4

The tail baseplate of bacteriophage T4 contains a structurally essential, three-domain protein encoded by gene 5 in which the middle domain possesses lysozyme activity. The gene 5 product (gp5) undergoes post-translational cleavage, allowing the resultant N-terminal domain (gp5*) to assemble into the baseplate as a trimer. The lysozyme activity of the undissociated cleaved gp5 is inhibited until infection has been initiated, when the C-terminal portion of the molecule is detached and the rest of the molecule dissociates into monomers. The 3D structure of the undissociated cleaved gp5, complexed with gp27 (another component of the baseplate), shows that it is a cell-puncturing device that functions to penetrate the outer cell membrane and to locally dissolve the periplasmic cell wall.

Crystallographic Analysis Reveals Octamerization of Viroplasm Matrix Protein P9-1 of Rice Black Streaked Dwarf Virus

ABSTRACT 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.

Dual modification of a triple-stranded β-helix nanotube with Ru and Re metal complexes to promote photocatalytic reduction of CO2

We have constructed a robust β-helical nanotube from the component proteins of bacteriophage T4 and modified this nanotube with Ru(II)(bpy)(3) and Re(I)(bpy)(CO)(3)Cl complexes. The photocatalytic system arranged on the tube catalyzes the reduction of CO(2) with higher reactivity than that of the mixture of the monomeric forms.

Construction of Robust Bio‐nanotubes using the Controlled Self‐Assembly of Component Proteins of Bacteriophage T4

of functional molecules at appropriate sites. Thus, it has remained challenging to obtain tube structures with high sta-bility and well-defi ned nanoscale lengths for properly aligning synthetic molecules on the surfaces of the nanotubes. The β -helical protein motif holds promise as a candidate to overcome these problems. This motif occurs predominantly in native tubular structural proteins such as antifreeze proteins, prions, and viruses.