Expression and Properties of Bacteriophage T4 Gene Product 11

A plasmid vector for expression of bacteriophage T4 gene product 11 (gp11) in E. coli cells has been constructed. Gp11 is a baseplate protein that connects short tail fibers providing irreversible adsorption of the virus on a cell. A method based on chromatography on hydroxyapatite has been developed for purification of recombinant gp11. The protein is active in an in vitro complementation assay and transforms defective phage particles lacking gp11 into infective ones. Gel filtration data suggest that the biologically active protein is a trimer. According to CD spectroscopy and sequence analysis data, the polypeptide chain of gp11 contains not less than 20% -helical segments, about 30% -structure, and belongs to the class of / structural proteins.     

Structure and folding of bacteriophage T4 gene product 9 triggering infection. I. Production and properties of recombinant protein

Gene product 9 (gp9, 288 amino acid residues per monomer, molecular weight 30.7 kD) of bacteriophage T4 triggers the baseplate reorganization and the sheath contraction after interaction of the long tail fibers with the receptors of the bacterial cell. In this work we have produced the recombinant protein and determined that gp9 is a stable homotrimer and active in in vitro complementation assay completing the defective phage particles which lack gp9. According to CD-spectroscopy data, the gp9 polypeptide chain contains 65-73% beta-structure and 11-16% alpha-helical segments, this being in good agreement with secondary structure prediction results. Additionally, we have constructed a set of plasmid vectors for expression of gp9 deletion mutants. The fragments with consecutive truncations of the N-terminus of the molecule, as well as the full-length protein, are trimers resistant to SDS treatment and decrease infective phage particle formation in in vitro complementation assay with native gp9. The deletion of the molecule C-terminal region results in failure of trimerization and decreases the stability of the protein.     

The structure of bacteriophage T4 gene product 9: the trigger for tail contraction.

BACKGROUND: The T4 bacteriophage consists of a head, filled with double-stranded DNA, and a complex contractile tail required for the ejection of the viral genome into the Escherichia coli host. The tail has a baseplate to wh?ch are attached six long and six short tail fibers. These fibers are the sensing devices for recognizing the host. When activated by attachment to cell receptors, the fibers cause a conformational transition in the baseplate and subsequently in the tail sheath, which initiates DNA ejection. The baseplate is a multisubunit complex of proteins encoded by 15 genes. Gene product 9 (gp9) is the protein that connects the long tail fibers to the baseplate and triggers the tail contraction after virus attachment to a host cell. RESULTS: The crystal structure of recombinant gp9, determined to 2.3 A resolution, shows that the protein of 288 amino acid residues assembles as a homotrimer. The monomer consists of three domains: the N-terminal domain generates a triple coiled coil; the middle domain is a mixed, seven-stranded beta sandwich with a topology not previously observed; and the C-terminal domain is an eight-stranded, antiparallel beta sandwich having some resemblance to 'jelly-roll' viral capsid protein structures. CONCLUSIONS: The biologically active form of gp9 is a trimer. The protein contains flexible interdomain hinges, which are presumably required to facilitate signal transmission between the long tail fibers and the baseplate. Structural and genetic analyses show that the C-terminal domain is bound to the baseplate, and the N-terminal coiled-coil domain is associated with the long tail fibers.     

Structure of the central hub of bacteriophage Mu baseplate determined by X-ray crystallography of gp44

Bacteriophage Mu is a double-stranded DNA phage that consists of an icosahedral head, a contractile tail with baseplate and six tail fibers, similar to the well-studied T-even phages. The baseplate of bacteriophage Mu, which recognizes and attaches to a host cell during infection, consists of at least eight different proteins. The baseplate protein, gp44, is essential for bacteriophage Mu assembly and the generation of viable phages. To investigate the role of gp44 in baseplate assembly and infection, the crystal structure of gp44 was determined at 2.1A resolution by the multiple isomorphous replacement method. The overall structure of the gp44 trimer is similar to that of the T4 phage gp27 trimer, which forms the central hub of the T4 baseplate, although these proteins share very little primary sequence homology. Based on these data, we confirm that gp44 exists as a trimer exhibiting a hub-like structure with an inner diameter of 25A through which DNA can presumably pass during infection. The molecular surface of the gp44 trimer that abuts the host cell membrane is positively charged, and it is likely that Mu phage interacts with the membrane through electrostatic interactions mediated by gp44.     

Structure and location of gene product 8 in the bacteriophage T4 baseplate

Many bacteriophages, such as T4, T7, RB49, and phi29, have complex, sometimes multilayered, tails that facilitate an almost 100% success rate for the viral particles to infect host cells. In bacteriophage T4, there is a baseplate, which is a multiprotein assembly, at the distal end of the contractile tail. The baseplate communicates to the tail that the phage fibers have attached to the host cell, thereby initiating the infection process. Gene product 8 (gp8), whose amino acid sequence consists of 334 residues, is one of at least 16 different structural proteins that constitute the T4 baseplate and is the sixth baseplate protein whose structure has been determined. A 2.0A resolution X-ray structure of gp8 shows that the two-domain protein forms a dimer, in which each monomer consists of a three-layered beta-sandwich with two loops, each containing an alpha-helix at the opposite sides of the sandwich. The crystals of gp8 were produced in the presence of concentrated chloride and bromide ions, resulting in at least 11 halide-binding sites per monomer. Five halide sites, situated at the N termini of alpha-helices, have a protein environment observed in other halide-containing protein crystal structures. The computer programs EMfit and SITUS were used to determine the positions of six gp8 dimers within the 12A resolution cryo-electron microscopy image reconstruction of the baseplate-tail tube complex. The gp8 dimers were found to be located in the upper part of the baseplate outer rim. About 20% of the gp8 surface is involved in contacts with other baseplate proteins, presumed to be gp6, gp7, and gp10. With the structure determination of gp8, a total of 53% of the volume of the baseplate has now been interpreted in terms of its atomic structure.     

Structure, stability, and biological activity of bacteriophage T4 gene product 9 probed with mutagenesis and monoclonal antibodies

Gene product (gp) 9 connects the long tail fibers and triggers the structural transition of T4 phage baseplate at the beginning of infection process. Gp9 is a parallel homotrimer with 288 amino acid residues per chain that forms three domains. To investigate the role of the gp9 amino terminus, we have engineered a set of mutants with deletions and random substitutions in this part. The structure of the mutants was probed using monoclonal antibodies that bind to either N-terminal, middle, or C-terminal domains. Deletions of up to 12 N-terminal residues as well as random substitutions of the second, third and fourth residues yielded trimers that failed to incorporate in vitro into the T4 9(-)-particles and were not able to convert them into infectious virions. As detected using monoclonal antibodies, these mutants undergo structural changes in both N-terminal and middle domains. Furthermore, deletion of the first twenty residues caused profound structural changes in all three gp9 domains. In addition, N-terminally truncated proteins and randomized mutants formed SDS-resistant "conformers" due to unwinding of the N-terminal region. Co-expression of the full-length gp9 and the mutant lacking first 20 residues clearly shows the assembly of heterotrimers, suggesting that the gp9 trimerization in vivo occurs post-translationally. Collectively, our data indicate that the aminoterminal sequence of gp9 is important to maintain a competent structure capable of incorporating into the baseplate, and may be also required at intermediate stages of gp9 folding and assembly.     

Structure and folding of bacteriophage T4 gene product 9 triggering infection. II. Study Of conformational changes of gene product 9 mutants using monoclonal antibodies

Gene product 9 (gp9) of bacteriophage T4, whose spatial structure we have recently solved to 2.3 A resolution, is a convenient model for studying the folding and oligomerization mechanisms of complex proteins. The gp9 polypeptide chain consists of 288 amino acids forming three domains. Three monomers, packed in parallel, assemble to a functionally active protein. The main aim of this work was to study conformational changes and trimerization of gp9 deletion mutants using monoclonal antibodies (mAbs). We selected a set of mAbs interacting with the amino, middle, and carboxyl regions of the protein, respectively. Eighteen mAbs bind to native as well as to denatured protein, and two mAbs bind to denatured protein only. Using mAbs, we found that deletions of the gp9 N-terminal region result in conformational changes in the middle and C-terminal domains. The study of mAb binding to the CDelta. truncated mutant by competitive ELISA and immunoblotting shows that the C-terminus of the gp9 sequence is essential for protein trimerization and stability. A single point substitution of the Gln282 residue causes formation of a labile trimer that has significant conformational changes in the protein domains. The results of our study show that folding and trimerization of gp9 is a cooperative process that involves all domains of the protein.     

Evolution of bacteriophage tails: Structure of T4 gene product 10

The success of tailed bacteriophages to infect cells far exceeds that of most other viruses on account of their specialized tail and associated baseplate structures. The baseplate protein gene product (gp) 10 of bacteriophage T4, whose structure was determined to 1.2 A resolution, was fitted into the cryo-electron microscopy structures of the pre and post-infection conformations of the virus. gp10 functions as a molecular lever that rotates and extends the hinged short tail fibers to facilitate cell attachment. The central folding motif of the gp10 trimer is similar to that of the baseplate protein gp11 and to the receptor-binding domain of the short tail fiber, gp12. The three proteins comprise the periphery of the baseplate and interact with each other. The structural and functional similarities of gp10, gp11, and gp12 and their sequential order in the T4 genome suggest that they evolved separately, subsequent to gene triplication from a common ancestor. Such events are usual in the evolution of complex organelles from a common primordial molecule.     

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.     

Optimization of human immunodeficiency virus type 1 envelope glycoproteins with V1/V2 deleted, using virus evolution

The human immunodeficiency virus type 1 envelope glycoprotein (Env) complex is the principal focus of neutralizing antibody-based vaccines. The functional Env complex is a trimer consisting of six individual subunits: three gp120 molecules and three gp41 molecules. The individual subunits have proven unsuccessful as vaccines presumably because they do not resemble the functional Env complex. Variable domains and carbohydrates shield vulnerable neutralization epitopes on the functional Env complex. The deletion of variable loops has been shown to improve gp120's immunogenicity; however, problems have been encountered when introducing such modifications in stabilized Env trimer constructs. To address these issues, we have created a set of V1/V2 and V3 loop deletion variants in the context of complete virus to allow optimization by forced virus evolution. Compensatory second-site substitutions included the addition and/or removal of specific carbohydrates, changes in the disulfide-bonded architecture of the V1/V2 stem, the replacement of hydrophobic residues by hydrophilic and charged residues, and changes in distal parts of gp120 and gp41. These viruses displayed increased sensitivity to neutralizing antibodies, demonstrating the improved exposure of conserved domains. The results show that we can select for functionally improved Env variants with loop deletions through forced virus evolution. Selected evolved Env variants were transferred to stabilized Env trimer constructs and were shown to improve trimer expression and secretion. Based on these findings, we can make recommendations on how to delete the V1/V2 domain from recombinant Env trimers for vaccine and X-ray crystallography studies. In general, virus evolution may provide a powerful tool to optimize Env vaccine antigens.     

Expression and characterization of a baseplate protein for bacteriophage Mu, gp44

The gene product of gene 44 of Mu phage (gp44) is an essential protein for baseplate assembly and has been designated as gpP, a traditional genetic assignment. The function of gp44 during the assembly or infection process is not known. In the present study, we purified the recombinant gp44 and characterized it by analytical ultracentrifugation and differential scanning microcalorimetry. The results indicate that gp44 forms a trimer comprising a complex consisting of the 42 kDa and 40 kDa subunits that had been cleaved in the C-terminal region. Thermodynamic analysis also suggested that the C-terminal region forms a flexible domain.     

Multiple enzymatic activities of the murein hydrolase from staphylococcal phage phi11. Identification of a D-alanyl-glycine endopeptidase activity.

Bacteriophage muralytic enzymes degrade the cell wall envelope of staphylococci to release phage particles from the bacterial cytoplasm. Murein hydrolases of staphylococcal phages phi11, 80alpha, 187, Twort, and phiPVL harbor a central domain that displays sequence homology to known N-acetylmuramyl-L-alanyl amidases; however, their precise cleavage sites on the staphylococcal peptidoglycan have thus far not been determined. Here we examined the properties of the phi11 enzyme to hydrolyze either the staphylococcal cell wall or purified cell wall anchor structures attached to surface protein. Our results show that the phi11 enzyme has D-alanyl-glycyl endopeptidase as well as N-acetylmuramyl-L-alanyl amidase activity. Analysis of a deletion mutant lacking the amidase-homologous sequence, phi11(Delta181-381), revealed that the D-alanyl-glycyl endopeptidase activity is contained within the N-terminal 180 amino acid residues of the polypeptide chain. Sequences similar to this N-terminal domain are found in the murein hydrolases of staphylococcal phages but not in those of phages that infect other Gram-positive bacteria such as Listeria or Bacillus.     

The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities

The TWINKLE protein is a hexameric DNA helicase required for replication of mitochondrial DNA. TWINKLE displays striking sequence similarity to the bacteriophage T7 gene 4 protein (gp4), which is a bi-functional primase-helicase required at the phage DNA replication fork. The N-terminal domain of human TWINKLE contains some of the characteristic sequence motifs found in the N-terminal primase domain of the T7 gp4, but other important motifs are missing. TWINKLE is not an active primase in vitro and the functional role of the N-terminal region has remained elusive. In this report, we demonstrate that the N-terminal part of TWINKLE is required for efficient binding to single-stranded DNA. Truncations of this region reduce DNA helicase activity and mitochondrial DNA replisome processivity. We also find that the gp4 and TWINKLE are functionally distinct. In contrast to the phage protein, TWINKLE binds to double-stranded DNA. Moreover, TWINKLE forms stable hexamers even in the absence of Mg²⁺ or NTPs, which suggests that an accessory protein, a helicase loader, is needed for loading of TWINKLE onto the circular mtDNA genome.     

Chaperonin-mediated folding of bacteriophage T4 major capsid protein. II. Production of gene product 23 deletion mutants

Folding of bacteriophage T4 major capsid protein, gene product 23 (534 a.a.), is aided by two proteins: E. coli GroEL chaperonin and viral gp31 co-chaperonin. In the present work a set of mutants with extensive deletions inside gene 23 using controlled digestion with Bal31 nuclease has been constructed. Proteins with deletions were co-expressed from plasmid vectors with phage gp31 co-chaperonin. Deletions from 8 to 33 a.a. in the N-terminal region of the gp23 molecule covering the protein proteolytic cleavage site during capsid maturation have no influence on the mutants' ability to produce in E. coli cells proteins which form regular structures—polyheads. Deletions in other regions of the polypeptide chain (187-203 and 367-476 a.a.) disturb the correct folding and subsequent assembly of gp23 into polyheads.     

The tailspike protein of Shigella phage Sf6. A structural homolog of Salmonella phage P22 tailspike protein without sequence similarity in the beta-helix domain

Bacteriophage Sf6 tailspike protein is functionally equivalent to the well characterized tailspike of Salmonella phage P22, mediating attachment of the viral particle to host cell-surface polysaccharide. However, there is significant sequence similarity between the two 70-kDa polypeptides only in the N-terminal putative capsid-binding domains. The major, central part of P22 tailspike protein, which forms a parallel beta-helix and is responsible for saccharide binding and hydrolysis, lacks detectable sequence homology to the Sf6 protein. After recombinant expression in Escherichia coli as a soluble protein, the Sf6 protein was purified to homogeneity. As shown by circular dichroism and Fourier transform infrared spectroscopy, the secondary structure contents of Sf6 and P22 tailspike proteins are very similar. Both tailspikes are thermostable homotrimers and resist denaturation by SDS at room temperature. The specific endorhamnosidase activities of Sf6 tailspike protein toward fluorescence-labeled dodeca-, deca-, and octasaccharide fragments of Shigella O-antigen suggest a similar active site topology of both proteins. Upon deletion of the N-terminal putative capsid-binding domain, the protein still forms a thermostable, SDS-resistant trimer that has been crystallized. The observations strongly suggest that the tailspike of phage Sf6 is a trimeric parallel beta-helix protein with high structural similarity to its functional homolog from phage P22.     

Identification of the HIV-1 gp41 core-binding motif in the scaffolding domain of caveolin-1

The human immunodeficiency virus, type 1 (HIV-1) gp41 core plays an important role in fusion between viral and target cell membranes. A single chain polypeptide, N36(L8)C34, which forms a six-helix bundle in physiological solution, can be used as a model of gp41 core. Here we identified from a 12-mer phage peptide library a positive phage clone displaying a peptide sequence with high binding activity to the HIV-1 gp41 core. The peptide sequence contains a putative gp41-binding motif, PhiXXXXPhiXPhi (X is any amino acid residue, and Phi is any one of the aromatic amino acid residues Trp, Phe, or Tyr). This motif also exists in the scaffolding domain of caveolin-1 (Cav-1), a known gp41-binding protein. Cav-1-(61-101) and Cav-1-(82-101), two recombinant fusion proteins containing the Cav-1 scaffolding domain, bound significantly to the gp41 expressed in mammalian cells and interacted with the polypeptide N36(L8)C34. These results suggest that the scaffolding domain of Cav-1 may bind to the gp41 core via the motif. This interaction may be essential for formation of fusion pore or endocytosis of HIV-1 and affect the pathogenesis of HIV-1 infection. Further characterization of the gp41 core-binding motifs may shed light on the alternative mechanism by which HIV-1 enters into the target cell.     

The helicase domain of phage P4 alpha protein overlaps the specific DNA binding domain.

Replication initiation depends on origin recognition, helicase, and primase activities. In phage P4, a second DNA region, the cis replication region (crr), is also required for replication initiation. The multifunctional alpha protein of phage P4, which is essential for DNA replication, combines the three aforementioned activities on a single polypeptide chain. Protein domains responsible for the activities were identified by mutagenesis. We show that mutations of residues G506 and K507 are defective in vivo in phage propagation and in unwinding of a forked helicase substrate. This finding indicates that the proposed P loop is essential for helicase activity. Truncations of gene product alpha (gp alpha) demonstrated that 142 residues of the C terminus are sufficient for specifically binding ori and crr DNA. The minimal binding domain retains gp alpha's ability to induce loop formation between ori and crr. In vitro and in vivo analysis of short C-terminal truncations indicate that the C terminus is needed for helicase activity as well as for specific DNA binding.     

Structure of the bacteriophage phi KZ lytic transglycosylase gp144

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage phi KZ has been determined to 2.5-A resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. phi KZ gp144 is a 260-residue alpha-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G D-Ala-D-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu 115 in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the phi KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine)(4) has been determined to 2.6-A resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.     

Structural and Functional Studies of gpX of Escherichia coli Phage P2 Reveal a Widespread Role for LysM Domains in the Baseplates of Contractile-Tailed Phages

A variety of bacterial pathogenicity determinants, including the type VI secretion system and the virulence cassettes from Photorhabdus and Serratia, share an evolutionary origin with contractile-tailed myophages. The well-characterized Escherichia coli phage P2 provides an excellent system for studies related to these systems, as its protein composition appears to represent the “minimal” myophage tail. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of gpX, a 68-residue tail baseplate protein. Although the sequence and structure of gpX are similar to those of LysM domains, which are a large family associated with peptidoglycan binding, we did not detect a peptidoglycan-binding activity for gpX. However, bioinformatic analysis revealed that half of all myophages, including all that possess phage T4-like baseplates, encode a tail protein with a LysM-like domain, emphasizing a widespread role for this domain in baseplate function. While phage P2 gpX comprises only a single LysM domain, many myophages display LysM domain fusions with other tail proteins, such as the DNA circulation protein found in Mu-like phages and gp53 of T4-like phages. Electron microscopy of P2 phage particles with an incorporated gpX-maltose binding protein fusion revealed that gpX is located at the top of the baseplate, near the junction of the baseplate and tail tube. gpW, the orthologue of phage T4 gp25, was also found to localize to this region. A general colocalization of LysM-like domains and gpW homologues in diverse phages is supported by our bioinformatic analysis.