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, 408 (6813), 745-50

Structure of the Bacteriophage phi29 DNA Packaging Motor

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Structure of the Bacteriophage phi29 DNA Packaging Motor

A A Simpson et al. Nature.

Abstract

Motors generating mechanical force, powered by the hydrolysis of ATP, translocate double-stranded DNA into preformed capsids (proheads) of bacterial viruses and certain animal viruses. Here we describe the motor that packages the double-stranded DNA of the Bacillus subtilis bacteriophage phi29 into a precursor capsid. We determined the structure of the head-tail connector--the central component of the phi29 DNA packaging motor--to 3.2 A resolution by means of X-ray crystallography. We then fitted the connector into the electron densities of the prohead and of the partially packaged prohead as determined using cryo-electron microscopy and image reconstruction analysis. Our results suggest that the prohead plus dodecameric connector, prohead RNA, viral ATPase and DNA comprise a rotary motor with the head-prohead RNA-ATPase complex acting as a stator, the DNA acting as a spindle, and the connector as a ball-race. The helical nature of the DNA converts the rotary action of the connector into translation of the DNA.

Figures

Figure 1
Figure 1
Cryo-EM reconstructions. Top and bottom rows: a, mature ϕ29; b, prohead + 120-base pRNA; c, prohead treated with RNase; d, difference map between b and c; and e, partially packaged particle (with DNA in channel). End-on views, looking along the tail towards the head, are given for b and e. Orthogonal difference pRNA densities are shown below the difference map in d. The arrow in a shows the position of the section below that was obtained by averaging particles without imposing five-fold symmetry. Note that both the five-fold symmetry of the downward-pointing head fibres (numbered 1 to 5) and six-fold symmetry (numbered 1 to 5) of the lower collar are visible.
Figure 2
Figure 2
Connector structure ribbon diagrams. a, The dodecameric connector seen from the tail looking towards the head; b, a side view with the pRNA-binding site at the bottom, showing the conical structure of the connector and the helical twist of each subunit around the 12-fold axis (white); c, a stereo diagram of a pair of monomers. One monomer is coloured red in the central domain, green in the wide-end domain that resides inside the capsid, and yellow at the narrow-end domain. The other monomers are all coloured blue. The ordered part of the polypeptide starts with helix α1 on the outside of the connector, going towards the wide end (residues 61 to 128 and 247 to 286). Helix α3 (residues 129 to 157) returns the chain to the narrow end (residues 158 to 202). The tip of the connector at the narrow end is formed by residues 164 to 170 and 185 to 196. Helix α5 (residues 208 to 226) returns the polypeptide to the wide end through the second disordered section. (Drawn with the program MOLSCRIPT and RASTER3D.)
Figure 3
Figure 3
Cryo-EM density fitted with atomic structures. a, Cross-section of the cryo-EM prohead density (red) fitted with the Cα backbone of the connector (yellow) and the cryo-EM pRNA difference map (green). Shown also is a DNA molecule placed through the central channel of the connector. The prohead capsid, one of the five contacts between the pRNA with the capsid, and the partially disordered residues 229 to 246 and 287 to 309 in the connector are indicated. b, Stereo drawing of the 120-base pRNA pentamer fitted into the cryo-EM density. Secondary structural elements are shown in white (A helix), blue (C helix), orange (E helix), yellow (CE loop), green (D helix) and red (D loop). Four intermolecular base pairs are shown in yellow-red. It had previously been shown that helices D, C and E participate in binding pRNA to the connector, and it was suggested that helix A binds the viral ATPase (gp16). (Drawn with the programs XTALVIEW and RASTER3D.)
Figure 4
Figure 4
The DNA packaging mechanism. Shown is one cycle in the mechanism that rotates the connector and translates the DNA into the head. The view down the connector axis (top) is towards the head, whereas the bottom row shows side views corresponding to that seen in Fig. 2b. Eleven of the 12 subunits (A, B,…, L) of the connector are shown in green; the ‘active’ monomer is shown in red. The connector is represented as a set of small spheres at the narrow end and a set of larger spheres at the wide end connected by a line representing the central helical region. The pRNA–ATPase complexes (I, II, III, IV, V), surrounding the narrow end, are shown by a set of four blue spheres and one red sphere. The DNA base aligned with the active connector monomer is also shown in red. In a, the active pRNA–ATPase I interacts with the adjacent connector monomer (A), which in turn contacts the aligned DNA base. In b, the narrow end of the connector has moved anticlockwise by 12° to place the narrow end of monomer C opposite ATPase II, the next ATPase to be fired, causing the connector to expand lengthwise by slightly changing the angle of the helices in the central domain (white arrow with asterisk). In c, the wide end of the connector has followed the narrow end, while the connector relaxes and contracts (white arrow with two asterisks), thus causing the DNA to be translated into the phage head. For the next cycle, ATPase II is activated, causing the connector to be rotated another 12°, and so forth. (Drawn with the program RASTER3D.)

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