Structural basis for DNA recognition and loading into a viral packaging motor

York Structural Biology Laboratory, Department of Chemistry, University of York, York, YO10 5DD, United Kingdom.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 12/2011; 109(3):811-6. DOI: 10.1073/pnas.1110270109
Source: PubMed

ABSTRACT Genome packaging into preformed viral procapsids is driven by powerful molecular motors. The small terminase protein is essential for the initial recognition of viral DNA and regulates the motor's ATPase and nuclease activities during DNA translocation. The crystal structure of a full-length small terminase protein from the Siphoviridae bacteriophage SF6, comprising the N-terminal DNA binding, the oligomerization core, and the C-terminal β-barrel domains, reveals a nine-subunit circular assembly in which the DNA-binding domains are arranged around the oligomerization core in a highly flexible manner. Mass spectrometry analysis and four further crystal structures show that, although the full-length protein exclusively forms nine-subunit assemblies, protein constructs missing the C-terminal β-barrel form both nine-subunit and ten-subunit assemblies, indicating the importance of the C terminus for defining the oligomeric state. The mechanism by which a ring-shaped small terminase oligomer binds viral DNA has not previously been elucidated. Here, we probed binding in vitro by using EPR and surface plasmon resonance experiments, which indicated that interaction with DNA is mediated exclusively by the DNA-binding domains and suggested a nucleosome-like model in which DNA binds around the outside of the protein oligomer.

Download full-text


Available from: Juan C Alonso, Aug 11, 2015
  • Source
    • "The relationships between these more complete structures to the fragment structures of l and phage 44RR2 TerS structures are less clear (but see Gao and Rao, 2011). Nonetheless, in all of the TerS structures the N-terminal domain is largely helical and includes a helix-turn-helix motif that may be the DNA-binding portion of these proteins (Buttner et al., 2012; Roy et al., 2012; Zhao et al., 2012; Sun et al., 2012). Analysis of mutations of Sf6 TerS have indicated that its N-terminal domain is responsible for binding DNA nonspecifically in vitro (Zhao et al., 2010, 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Bacteriophage Sf6 DNA packaging series initiate at many locations across a 2kbp region. Our in vivo studies show that Sf6 small terminase subunit (TerS) protein recognizes a specific packaging (pac) site near the center of this region, that this site lies within the portion of the Sf6 gene that encodes the DNA-binding domain of TerS protein, that this domain of the TerS protein is responsible for the imprecision in Sf6 packaging initiation, and that the DNA-binding domain of TerS must be covalently attached to the domain that interacts with the rest of the packaging motor. The TerS DNA-binding domain is self-contained in that it apparently does not interact closely with the rest of the motor and it binds to a recognition site that lies within the DNA that encodes the domain. This arrangement has allowed the horizontal exchange of terS genes among phages to be very successful.
    Virology 04/2013; 440(2). DOI:10.1016/j.virol.2013.02.023 · 3.28 Impact Factor
  • Source
    • "Although octamers were also observed in solution for bacteriophage T7 (gp18) (White and Richardson, 1987) and T4 (gp16) (Lin et al., 1997) S-terminases, a recent crystal structure of the T4-like phage 44RR S-terminase (gp16) revealed a mix of undecamers and dodecamers (Sun et al., 2012). Similarly, phage SPP1 S-terminase (gp1) was reported to form decameric rings in solution (Camacho et al., 2003), but the crystal structure of the SPP1-like Bacillus phage SF6 revealed a mix of nonamers and decamers, with the nonamer being the predominant conformation (Bü ttner et al., 2012). Finally, phage l S-terminase (gpNu1) forms a hetero-trimer bound to a monomer of L-terminase (gpA1), and in vitro this protomer can further assemble into tetramers (Maluf et al., 2006). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Packaging of viral genomes into empty procapsids is powered by a large DNA-packaging motor. In most viruses, this machine is composed of a large (L) and a small (S) terminase subunit complexed with a dodecamer of portal protein. Here we describe the 1.75 Å crystal structure of the bacteriophage P22 S-terminase in a nonameric conformation. The structure presents a central channel ∼23 Å in diameter, sufficiently large to accommodate hydrated B-DNA. The last 23 residues of S-terminase are essential for binding to DNA and assembly to L-terminase. Upon binding to its own DNA, S-terminase functions as a specific activator of L-terminase ATPase activity. The DNA-dependent stimulation of ATPase activity thus rationalizes the exclusive specificity of genome-packaging motors for viral DNA in the crowd of host DNA, ensuring fidelity of packaging and avoiding wasteful ATP hydrolysis. This posits a model for DNA-dependent activation of genome-packaging motors of general interest in virology.
    Structure 07/2012; 20(8):1403-13. DOI:10.1016/j.str.2012.05.014 · 6.79 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Tailed DNA bacteriophages assemble empty procapsids that are subsequently filled with the viral genome by means of a DNA packaging machine situated at a special fivefold vertex. The packaging machine consists of a "small terminase" and a "large terminase" component. One of the functions of the small terminase is to initiate packaging of the viral genome, whereas the large terminase is responsible for the ATP-powered translocation of DNA. The small terminase subunit has three domains, an N-terminal DNA-binding domain, a central oligomerization domain, and a C-terminal domain for interacting with the large terminase. Here we report structures of the central domain in two different oligomerization states for a small terminase from the T4 family of phages. In addition, we report biochemical studies that establish the function for each of the small terminase domains. On the basis of the structural and biochemical information, we propose a model for DNA packaging initiation.
    Proceedings of the National Academy of Sciences 12/2011; 109(3):817-22. DOI:10.1073/pnas.1110224109 · 9.81 Impact Factor
Show more