Structure of the receptor-binding protein of bacteriophage det7: a podoviral tail spike in a myovirus.

A new Salmonella enterica phage, Det7, was isolated from sewage and shown by electron microscopy to belong to the Myoviridae morphogroup of bacteriophages. Det7 contains a 75-kDa protein with 50% overall sequence identity to the tail spike endorhamnosidase of podovirus P22. Adsorption of myoviruses to their bacterial hosts is normally mediated by long and short tail fibers attached to a contractile tail, whereas podoviruses do not contain fibers but attach to host cells through stubby tail spikes attached to a very short, noncontractile tail. The amino-terminal 150 residues of the Det7 protein lack homology to the P22 tail spike and are probably responsible for binding to the base plate of the myoviral tail. Det7 tail spike lacking this putative particle-binding domain was purified from Escherichia coli, and well-diffracting crystals of the protein were obtained. The structure, determined by molecular replacement and refined at a 1.6-A resolution, is very similar to that of bacteriophage P22 tail spike. Fluorescence titrations with an octasaccharide suggest Det7 tail spike to bind its receptor lipopolysaccharide somewhat less tightly than the P22 tail spike. The Det7 tail spike is even more resistant to thermal unfolding than the already exceptionally stable homologue from P22. Folding and assembly of both trimeric proteins are equally temperature sensitive and equally slow. Despite the close structural, biochemical, and sequence similarities between both proteins, the Det7 tail spike lacks both carboxy-terminal cysteines previously proposed to form a transient disulfide during P22 tail spike assembly. Our data suggest receptor-binding module exchange between podoviruses and myoviruses in the course of bacteriophage evolution.

Crystal structure of the avian reovirus inner capsid protein sigmaA.

Avian reovirus, an important avian pathogen, expresses eight structural and four nonstructural proteins. The structural sigmaA protein is a major component of the inner capsid, clamping together lambdaA building blocks. sigmaA has also been implicated in the resistance of avian reovirus to the antiviral action of interferon by strongly binding double-stranded RNA in the host cell cytoplasm and thus inhibiting activation of the double-stranded RNA-dependent protein kinase. We have solved the structure of bacterially expressed sigmaA by molecular replacement and refined it using data to 2.3-A resolution. Twelve sigmaA molecules are present in the P1 unit cell, arranged as two short double helical hexamers. A positively charged patch is apparent on the surface of sigmaA on the inside of this helix and mutation of either of two key arginine residues (Arg155 and Arg273) within this patch abolishes double-stranded RNA binding. The structural data, together with gel shift assay, electron microscopy, and sedimentation velocity centrifugation results, provide evidence for cooperative binding of sigmaA to double-stranded RNA. The minimal length of double-stranded RNA required for sigmaA binding was observed to be 14 to 18 bp.

Crystallization of the avian reovirus double-stranded RNA-binding and core protein sigmaA.

The avian reovirus protein sigmaA plays a dual role: it is a structural protein forming part of the transcriptionally active core, but it has also been implicated in the resistance of the virus to interferon by strongly binding double-stranded RNA and thus inhibiting the double-stranded RNA-dependent protein kinase. The sigmaA protein has been crystallized from solutions containing ammonium sulfate at pH values around 6. Crystals belonging to space group P1, with unit-cell parameters a = 103.2, b = 129.9, c = 144.0 A, alpha = 93.8, beta = 105.1, gamma = 98.2 degrees were grown and a complete data set has been collected to 2.3 A resolution. The self-rotation function suggests that sigmaA may form symmetric arrangements in the crystals.

Structure of the carboxy-terminal receptor-binding domain of avian reovirus fibre sigmaC.

Avian reovirus fibre, a homo-trimer of the sigmaC protein, is responsible for primary host cell attachment. The protein expressed in bacteria forms elongated fibres comprised of a carboxy-terminal globular head domain and a slender shaft, and partial proteolysis yielded a carboxy-terminal protease-stable domain that was amenable to crystallisation. Here, we show that this fragment retains receptor-binding capability and report its structure, solved using two-wavelength anomalous diffraction and refined using data collected from three different crystal forms at 2.1 angstroms, 2.35 angstroms and 3.0 angstroms resolution. The carboxy-terminal globular domain has a beta-barrel fold with the same overall topology as the mammalian reovirus fibre (sigma1). However, the monomers of the sigmaC trimer show a more splayed-out arrangement than in the sigma1 structure. Also resolved are two triple beta-spiral repeats of the shaft or stalk domain. The presence in the sequence of heptad repeats amino-terminal to these triple beta-spiral repeats suggests that the unresolved portion of the shaft domain contains a triple alpha-helical coiled-coil structure. Implications for the function and stability of the sigmaC protein are discussed.

Crystallization of the C-terminal globular domain of avian reovirus fibre.

Avian reovirus fibre, a homotrimer of the sigmaC protein, is responsible for primary host-cell attachment. Using the protease trypsin, a C-terminal sigmaC fragment containing amino acids 156-326 has been generated which was subsequently purified and crystallized. Two different crystal forms were obtained, one grown in the absence of divalent cations and belonging to space group P6(3)22 (unit-cell parameters a = 75.6, c = 243.1 A) and one grown in the presence of either zinc or cadmium sulfate and belonging to space group P321 (unit-cell parameters a = 74.7, c = 74.5 A and a = 73.1, c = 69.9 A for the Zn(II)- and Cd(II)-grown crystals, respectively). The first crystal form diffracted synchrotron radiation to 3.0 A resolution and the second form to 2.2-2.3 A. Its closest related structure, the C-terminal fragment of mammalian reovirus fibre, has only 18% sequence identity and molecular-replacement attempts were unsuccessful. Therefore, a search is under way for suitable heavy-atom derivatives and attempts are being made to grow protein crystals containing selenomethionine instead of methionine.