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: 9.67). 12/2011; 109(3):811-6. DOI: 10.1073/pnas.1110270109
Source: PubMed


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.

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    • "Viral packaging motors are fundamental molecular machines that power the delivery of viral genomes into preformed procapsid shells. Despite growing interest in this field of biology [51], a plethora of individual S-terminase and L-terminase subunit crystal structures [13] [16] [18] [20] [22] [23] [25] and recent advances in single-molecule biophysical analysis of packaging motors [52], not even moderate resolution information exists for a terminase complex. "
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    ABSTRACT: Packaging of viral genomes inside empty procapsids is driven by a powerful ATP-hydrolyzing motor, formed in many double strand DNA (dsDNA)-viruses by a complex of a small (S-terminase) and a large terminase (L-terminase) subunit, transiently docked at the portal vertex during genome-packaging. Despite recent progress in elucidating the structure of individual terminase subunits and their domains, little is known about the architecture of an assembled terminase complex. Here, we describe a bacterial co-expression system that yields milligram quantities of the S:L-terminase complex of the Salmonella-phage P22. In vivo assembled terminase complex was affinity-purified and stabilized by addition of non-hydrolysable ATP, which binds specifically to the ATPase domain of L-terminase. Mapping studies revealed the N-terminus of L-terminase ATPase domain (residues 1-58) contains a minimal S-terminase Binding Domain (SBD) sufficient for stoichiometric association with residues 140-162 of S-terminase, the L-terminase Binding Domain (LBD). Hydrodynamic analysis by analytical ultracentrifugation sedimentation velocity and native mass spectrometry revealed the purified terminase complex consists predominantly of one copy of the nonameric S-terminase bound to two equivalents of L-terminase (1S-terminase:2L-terminase). Direct visualization of this molecular assembly in negative stained micrographs yielded a three-dimensional asymmetric reconstruction that resembles a 'nutcracker' with two L-terminase protomers projecting from the C-termini of a S-terminase ring. This is the first direct visualization of a purified viral terminase complex analyzed in the absence of DNA and procapsid. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Aug 2015 · Journal of Molecular Biology
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    • ", and for the side-byside double T4 TerS protein only rings, see Fig. 7 in Lin et al. (1997)], and whether DNA moves through a central ring channel and/or wraps around the ring is debated (Buttner et al., 2012; Roy et al., 2012; Roy and Cingolani, 2012; Sun et al., 2012; Teschke, 2012; Zhao et al., 2010). Even more importantly, the relevance of the rings to function remains problematic since the single planar rings of variable numbers of monomers from different pac site phages have been produced at high protein expression levels or observed under crystallization conditions. "
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    ABSTRACT: DNA packaging into empty viral procapsids by ATP-driven motor proteins applies widely among viruses. Recent fluorescence studies of phage T4 reveal: 1) the small terminase subunit (TerS) synapses pac homologs by a twin ring mechanism to gauge DNA maturation and allow packaging by the large terminase subunit (TerL); 2) translocation of linear DNA is efficient by TerL acting alone; expansion of the procapsid is controlled by the portal-terminase assembly; 3) both ends of the packaged DNA are held at the portal, showing a loop of DNA is packaged; 4) transient spring-like compression of B form to A form-like DNA accompanies translocation; 5) the C-terminal domain of TerL is docked to the portal and moves toward it when stalled; 6) a portal bound resolvase can release stalled Y-DNA compression and allow translocation in vitro; and 7) ATP powered translocation on A form dsDNA is supported by recent hexameric helicase studies. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Feb 2015 · Virology
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    • "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). "
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    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.
    Full-text · Article · Apr 2013 · Virology
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