Journal of Molecular Biology
The X-Ray Crystal Structure of the Phage λ Tail Terminator Protein Reveals the Biologically Relevant Hexameric Ring Structure and Demonstrates a Conserved Mechanism of Tail Termination among Diverse Long-Tailed Phages
Introduction
The majority of bacteriophages possess icosahedral double-stranded-DNA-filled heads and long (∼ 50–200 nm) tails,1 which are crucial for host cell recognition, cell wall penetration, and viral genome delivery. These long-tailed phages can be divided into two distinct groups: those with contractile tails (Myoviridae) and those with non-contractile tails (Siphoviridae).2 In the contractile-tailed phages, a tail sheath protein surrounds the tail tube and contracts upon infection, allowing the tail tube to penetrate the host cell. In contrast, non-contractile tails lack a sheath and do not change shape significantly upon infection. Since phage tails comprise a complex arrangement of multiple copies of many different proteins that must interact in a precisely ordered pathway, elucidation of the mechanisms by which tails assemble has presented a fascinating challenge.
One issue pertaining to tail assembly that has attracted considerable attention over the years is the regulation of length.3, 4 Within any particular species of phage, virtually every particle possesses a tail with precisely the same length. The exact length of both contractile and non-contractile tails is set by a tape measure protein (TMP) where the tail length is proportional to the length of the TMP.5, 6, 7 However, an additional protein, known as the tail terminator protein (TrP), is required to prevent aberrant polymerization of the tail tube protein beyond the length encoded by the TMP (Fig. 1). The product of the bacteriophage λ U gene (gpU) is the best characterized TrP. In the absence of gpU, λ infections result in the formation of normal heads, which are produced in a pathway separate from tail assembly, and extremely elongated tails, which result from the uncontrolled polymerization of the tail tube protein gpV. In the completed tail structure, gpU assembles at the top of the tail tube,8 thereby terminating gpV polymerization and providing the surface for interaction with the head.9 gpU is presumed to constitute a hexameric ring within the tail structure because it spontaneously forms such rings in the presence of Mg2+ that match the size of the hexameric rings of gpV that comprise the bulk of the tail tube.8 However, prior to tail assembly, gpU remains monomeric even at millimolar concentrations, which allowed us to solve the solution structure of this form of the protein using NMR spectroscopy.10
Since all long-tailed phages must avoid uncontrolled tail tube polymerization, it is expected that they all encode a protein with tail terminating activity. Consistent with this notion, mutations that led to elongated tails similar to those observed in cells infected by phage λ U mutants have been identified in the contractile-tailed phages Mu, P2, T4, and SPO1.8, 10, 11, 12, 13, 14 However, the question of whether all of these phages use the same mechanism for tail termination remains open because their putative TrPs have not been characterized and no sequence similarity can be detected among them. An indication that the structure of TrPs may be conserved in diverse phages was provided by a strong similarity detected between the structure of gpU and the structure of protein STM4215 [Protein Data Bank (PDB) ID: 2GJV] encoded in a prophage element of Salmonella typhimurium.10 Although this prophage element is uncharacterized, the gene encoding STM4215 lies adjacent to other genes encoding components of a contractile tail, suggesting that it may be a TrP. Interestingly, STM4215 crystallized as a hexameric ring with dimensions similar to the ring of gpV seen in the λ tail, which strengthens the notion that it is indeed a TrP. Nevertheless, without knowledge of the biologically relevant quaternary structure of a bona fide TrP, it is impossible to arrive at definitive conclusions pertaining to the function of STM4215 and the potential for conservation of TrP structure and function among diverse phages.
In the current work, we have pursued further studies on phage λ gpU. Despite its relatively small size (14.8 kDa), this protein presents an intriguing object for investigation, possessing surfaces to mediate self-interaction and binding to both the phage head and tail. In addition, gpU multimerizes upon contact with the appropriate tail assembly intermediate, yet remains monomeric at high protein concentrations on its own. Knowledge of its biologically relevant quaternary structure is essential to fully understand the mechanisms by which gpU performs its functions. As is described here, we have solved a hexameric structure of gpU using X-ray crystallography. Although the formation of this structure was induced by crystallization, we have employed bioinformatic and mutagenesis studies to show that this structure is biologically relevant and have also identified the functional surfaces of gpU. Using this structure as a starting point, we performed further bioinformatic investigation to trace the evolutionary relationships between gpU and diverse TrPs from contractile-tailed phages. This work complements recently published work from our laboratory showing that the tail tube proteins of contractile and non-contractile phages are likely to be evolutionarily related.15
Section snippets
The X-ray crystal structure of gpU
Even though we had already determined the tertiary structure of gpU using NMR spectroscopy,10 we initiated crystallization studies on this protein with the hope that the conditions of crystallization would induce formation of a biologically relevant multimer as has been observed in other systems.16, 17 Since diffraction-quality crystals of gpU wild type (gpU-WT) were not obtained initially, we also tested mutants that had been investigated previously.10 Fortunately, diffraction-quality crystals
Discussion
In this work, we used X-ray crystallography to determine the structure of an oligomeric form of gpU, the TrP of phage λ. Although the oligomers obtained in this study were induced through the process of crystallization, we have strong evidence that they are biologically relevant and not an artifact of crystallization. The dimensions of hexameric gpU-D74A structure (∼ 84 Å in diameter with a 32-Å central pore) match closely to the dimensions observed for hexameric gpV in the λ phage tail (∼ 90 Å
Sample preparation and crystallization
The U gene was expressed fused to an N-terminal hexahistidine tag using the pET15b vector (Novagen) as reported previously.10 Protein expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to a final concentration of 1 mM followed by an overnight incubation at 25 °C. Cells were harvested by centrifugation (5000 rpm, 15 min), lysed by sonication in 10 mM imidazole, 300 mM NaCl, and 50 mM NaH2PO4·H2O (pH 8), and the cellular debris was removed by centrifugation
Acknowledgements
The authors gratefully thank Dante Neculai, Mirela Neculai, Shao-Yang Ku, and G. David Smith for their crystallographic assistance and many helpful discussions. We also thank Matthew Cordes for aid in detecting transitive homology among TrPs. Funding for this research was supported by an operating grant from the Canadian Institutes of Health Research to A.R.D. (Fund Number MOP-77680) and P.L.H. (Fund Number MT13337). P.L.H. is the recipient of a Canada Research Chair. Beam line X12C at the
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