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

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.bacteriophage SPP1 | DNA packaging | virus assembly | X-ray crystallography T he virus genome in tailed dsDNA bacteriophages and in the evolutionarily related herpes viruses is packaged into a preformed empty procapsid (1-3). A powerful ATP-fueled molecular machine drives the DNA with a speed of up to 1;800 bp∕s through the portal protein embedded in a unique vertex of the icosahedral procapsid (2-4). The molecular motor usually consists of the small and large terminase proteins. The small terminase plays a dual role in virus particle assembly: It (i) recognizes viral DNA during the initiation of packaging and (ii) modulates the ATPase and nuclease activities of the large terminase during DNA translocation (5, 6). After filling a procapsid, the rest of the DNA is then docked to the portal entrance of another procapsid where the process of DNA translocation is repeated (7).X-ray structures have been determined for portal proteins from bacteriophages φ29 (8), SPP1 (9), and P22 (10) and also for large terminases from bacteriophages T4 (6), RB49 (6), and SPP1 (11). Three-dimensional information on small terminases is limited to the cryo-EM structure of phage P22 small terminase (12), the NMR structure of the DNA-binding domain of phage λ gpNu1 (13), and the crystal structure of phage Sf6 small terminase (14). In the absence of accurate three-dimensional data for all three motor components of one particular phage, mapping of functional information to the structure and modeling molecular interactions between individual components is challenging. We have addressed this issue by extending the structural information on Bacillus subtilis bacteriophages SPP1 and SF6, two very closely related viruses of the Siphovi...

The putative small terminase from the thermophilic dsDNA bacteriophage G20C is a nine-subunit oligomer

“…Although both SEC-MALLS and crystallographic data indicate nine-subunit oligomers, as observed for the P22 and SF6 small terminases (Roy et al, 2012;Bü ttner et al, 2012), structure determination by molecular replacement is not possible owing to a complete lack of sequence similarity. The next stage of this project will focus on experimental phasing.…”

“…Here, we report the production of recombinant protein in Escherichia coli, protein purification and crystallization. The results of size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) and crystal data indicate that the protein forms ninesubunit oligomers, like the small terminases found in bacteriophages SF6 and P22 (Bü ttner et al, 2012;Roy et al, 2012).…”

“…All were shown to assemble into ring-like structures composed of 8-12 subunits arranged symmetrically around a central axis. The main topological domains identified in the small terminase are (i) the C-terminal oligomerization domain, which establishes inter-subunit contacts around a central tunnel, and (ii) the exposed N-terminal domain, which in phages SPP1 and SF6 binds to the recognition pac site DNA (Bü ttner et al, 2012). Interestingly, in phage P22 the DNA-binding function is attributed to a short segment at the C-terminus (Roy et al, 2012).…”

“…As mentioned above, there appear to be differences in the way that different terminases recognize the genomic DNA (Bü ttner et (Roy et al, 2012;Bü ttner et al, 2012).…”

“…Three-dimensional structural data are available for oligomeric assemblies of small terminases from several viruses including Sf6, SF6, T4 and P22 (Zhao et al, 2010;Bü ttner et al, 2012;Sun et al, 2012;Roy et al, 2012). All were shown to assemble into ring-like structures composed of 8-12 subunits arranged symmetrically around a central axis.…”