The tail structure of bacteriophage T4 and its mechanism of contraction

Bacteriophage T4 and related viruses have a contractile tail that serves as an efficient mechanical device for infecting bacteria. A three-dimensional cryo-EM reconstruction of the mature T4 tail assembly at 15-A resolution shows the hexagonal dome-shaped baseplate, the extended contractile sheath, the long tail fibers attached to the baseplate and the collar formed by six whiskers that interact with the long tail fibers. Comparison with the structure of the contracted tail shows that tail contraction is associated with a substantial rearrangement of the domains within the sheath protein and results in shortening of the sheath to about one-third of its original length. During contraction, the tail tube extends beneath the baseplate by about one-half of its total length and rotates by 345 degrees , allowing it to cross the host's periplasmic space.     

Primary structure of the tail sheath protein of bacteriophage T4 and its gene

Complete sequence determination of gene 18 encoding the tail sheath protein was carried out mainly by the Maxam-Gilbert method. Approximately 40 peptides contained in a tryptic digest and a lysyl endopeptidase digest of gp 18 were isolated by reversed-phase high-performance liquid chromatography. All the peptides were identified along the nucleotide sequence of gene 18 based on the amino acid compositions. These peptides cover 88% of the total primary structure. Furthermore, the amino acid sequences of 9 of the 40 peptides were determined by a gas-phase protein sequencer; one of them turned to be the N-terminal one. The C-terminal peptide in the tryptic digest was isolated from the unadsorbed fraction of affinity chromatography on immobilized anhydrotrypsin and the amino acid sequence was also determined. Thus, the complete primary structure of gp 18 was determined; it has 658 amino acid residues and a molecular weight of 71,160.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Molecular assembly and structure of the bacteriophage T4 tail

The tail of bacteriophage T4 undergoes large structural changes upon infection while delivering the phage genome into the host cell. The baseplate is located at the distal end of the contractile tail and plays a central role in transmitting the signal to the tail sheath that the tailfibers have been adsorbed by a host bacterium. This then triggers the sheath contraction. In order to understand the mechanism of assembly and conformational changes of the baseplate upon infection, we have determined the structure of an in vitro assembled baseplate through the three-dimensional reconstruction of cryo-electron microscopy images to a resolution of 3.8 Å from electron micrographs. The atomic structure was fitted to the baseplate structure before and after sheath contraction in order to elucidate the conformational changes that occur after bacteriophage T4 has attached itself to a cell surface. The structure was also used to investigate the protease digestion of the assembly intermediates and the mutation sites of the tail genes, resulting in a number of phenotypes.     

The tail sheath structure of bacteriophage T4: a molecular machine for infecting bacteria

The contractile tail of bacteriophage T4 is a molecular machine that facilitates very high viral infection efficiency. Its major component is a tail sheath, which contracts during infection to less than half of its initial length. The sheath consists of 138 copies of the tail sheath protein, gene product (gp) 18, which surrounds the central non‐contractile tail tube. The contraction of the sheath drives the tail tube through the outer membrane, creating a channel for the viral genome delivery. A crystal structure of about three quarters of gp18 has been determined and was fitted into cryo‐electron microscopy reconstructions of the tail sheath before and after contraction. It was shown that during contraction, gp18 subunits slide over each other with no apparent change in their structure.     

Nucleotide sequence of the tail sheath gene of bacteriophage T4 and amino acid sequence of its product.

The nucleotide sequence of gene 18 of bacteriophage T4 was determined by the Maxam-Gilbert method, partially aided by the dideoxy method. To confirm the deduced amino acid sequence of the tail sheath protein (gp18) that is encoded by gene 18, gp18 was extensively digested by trypsin or lysyl endopeptidase and subjected to reverse-phase high-performance liquid chromatography. Approximately 40 peptides, which cover 88% of the primary structure, were fractionated, the amino acid compositions were determined, and the corresponding sequences in DNA were identified. Furthermore, the amino acid sequences of 10 of the 40 peptides were determined by a gas phase protein sequencer, including N- and C-terminal sequences. Thus, the complete amino acid sequence of gp18, which consists of 658 amino acids with a molecular weight of 71,160, was determined.     

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.     

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.     

Assembly of the tail of bacteriophage T4

The protein products of at least 21 phage genes are needed for the formation of the tail of bacteriophage T4. Cells infected with amber mutants defective in these genes are blocked in the assembly process. By characterizing the intermediate structures and unassembled proteins accumulating in mutant-infected cells, we have been able to delineate most of the gene-controlled steps in tail assembly. Both the organized structures and unassembled proteins serve as precursors for in vitro tail assembly. We review here studies on the initiation, polymerization, and termination of the tail tube and contractile sheath and the genetic control of these processes. These studies make clear the importance of the baseplate; if baseplate formation is blocked (by mutation) the tube and sheath subunits remain essentially unaggregated, in the form of soluble subunits. Seventeen of the 21 tail genes specify proteins involved in baseplate assembly. The genes map contiguously in two separate clusters, one of nine genes and the other of eight genes. Recent studies show that the hexagonal baseplate is the end-product of two independent subassembly pathways. The proteins of the first gene cluster interact to form a structure which probably represents one-sixth of the outer radius. The products of the other gene cluster interact to form the central part of the baseplate. Most of the phage tail precursor proteins appear to be synthesized in a non-aggregating form; they are converted to a reactive form upon incorporation into preformed substrate complexes, without proteolytic cleavage. Thus reactive sites are limited to growing structures.     

The wac gene product of bacteriophage T4 contains coiled-coil structural patterns

The bacteriophage T4 late gene wac (whisker antigen control) encodes the protein which forms the fibrous structure on the neck of the virion called whiskers. Amino acid sequence analysis of wac gene product, as deduced from the nucleotide sequence, indicate ten alpha-helical domains (19-40 residues long) with coiled-coil structural patterns. These regions comprise about 70% of the entire 486 amino acid sequence. The alpha-helices are separated by short stretches of polypeptide chain which are similar to the loop regions of the globular protein sequences. We propose a structural model for the dimer of wac gene product molecule, that we call fibritin in which two polypeptide chains associate in a parallel fashion and form a segmented alpha-helical coiled-coil rod similar to epidermal keratins.     

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.