| dc.description.abstract | Sf6 is a double-stranded DNA (dsDNA) bacteriophage with a short, non-contractile tail. The tail is a sophisticated molecular machine made of 39 copies of four gene products, including the dodecameric tail adaptor gp7, the hexameric tail nozzle gp8, the trimeric tail needle gp9 and 6 copies of the trimeric tail spike gp14. It has been shown that the tail assembly occurs in a sequential manner. Here we report the high-resolution structure of the Sf6 tail adaptor protein gp7. Comparative structural studies reveal that during tail assembly the gp7 N-terminus undergoes structural rearrangement by repositioning two consecutive repeats of a conserved octad sequence motif, turning the molecule from the preassembly state to the postassembly state, which creates the binding site for the next tail component to attach to. These results provide a structural basis for a mechanism of sequence motifs repositioning by which the adaptor protein mediates the sequential assembly of the phage tail. Tail nozzle gp8 is the following component attached to gp7 in the tail. It is highly conserved between Sf6 and P22, but the structure is not known yet. Here, we did Small-Angle X-ray Scattering (SAXS) analysis on gp8 monomer, showing a brick-shaped, globular protein with a small protrusion. Fitting of the SAXS model into the electron cryo-microscopy (cryoEM) map of the entire tail machine has aided in defining molecular boundaries between gp8 monomers and neighboring subunits of other tail components. One of the important functions of the tail is to deliver viral DNA through host envelope to establish infection. Given the fact that the tail is too short to directly span bacterial envelope, it is assumed that during infection the short tail is extended by three DNA-injection proteins, gp11, gp12, and gp13, to drill through the three-layer envelope of the host cell to inject phage DNA into the host cytoplasm. We achieved the 3D EM reconstruction of gp12 decamer, revealing a tube-like assembly with a constricted channel presumably for dsDNA delivery. We then solved the X-ray structure of the gp12 N-terminal domain (gp12NTD) which, surprisingly, assembles into a undecamer in crystals. This 2.8Å gp12NTD structure represents the first high-resolution structure of tailed virus DNA-injection proteins. The gp12NTD molecule consists of eight α-helices, seven of which forms two helix bundles. Biochemical study suggests that the helix α8 is dispensable for gp12 homo-oligomerization. Analysis on the tertiary structure and the locations of Gly and Pro residues, for the first time, provides experimental foundation for the assumption that internal proteins are partially unfolded when travelling through the narrow tail channel. We also show that P22-gp20 (Sf6-gp12 ortholog) NTD has a highly similar structure and it also assembles to a undecamer. By analyzing the structure characteristics and the conserved features between Sf6-gp12 and P22-gp20, we discussed the possible scenario of gp12/gp20 travelling through tail channel. The gp12CTD is monomeric in solution, and the C-terminal 27 residues are essential for gp12:gp13 interaction. The stoichiometry of gp12 and gp13 is likely to be 1:1. Similarly, gp20 binds with gp16 (counterpart of gp13). Our work sheds light on the roles of the two DNA-injection proteins (Sf6-gp12/P22-gp20 and Sf6-gp13/P22-gp16) in assembly of the extended tail for DNA delivery. A high-resolution X-ray structure of the Non-Structural protein 1 N-terminal domain (NS1N) of Minute Virus of Mice (MVM) is also reported here as an effort to study DNA replication in parvovirus. MVM has a single-stranded DNA (ssDNA) genome with the two ends folding back to from double-stranded hetero-telomeres, providing origin of replication (Ori). NS1N binds to Ori to perform a series of functions including ssDNA nicking. The NS1N structure here shows potential sites for dsDNA binding, ssDNA binding and cleavage on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases. Metal derivative crystal structures reveal the nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. | |
