DNA Conformational Changes Play a Force-Generating Role during Bacteriophage Genome Packaging

Abstract

Motors that move DNA, or that move along DNA, play essential roles in DNA replication, transcription, recombination, and chromosome segregation. The mechanisms by which these DNA translocases operate remain largely unknown. Some double-stranded DNA (dsDNA) viruses use an ATP-dependent motor to drive DNA into preformed capsids. These include several human pathogens as well as dsDNA bacteriophages-viruses that infect bacteria. We previously proposed that DNA is not a passive substrate of bacteriophage packaging motors but is instead an active component of the machinery. We carried out computational studies on dsDNA in the channels of viral portal proteins, and they reveal DNA conformational changes consistent with that hypothesis. dsDNA becomes longer ("stretched") in regions of high negative electrostatic potential and shorter ("scrunched") in regions of high positive potential. These results suggest a mechanism that electrostatically couples the energy released by ATP hydrolysis to DNA translocation: The chemical cycle of ATP binding, hydrolysis, and product release drives a cycle of protein conformational changes. This produces changes in the electrostatic potential in the channel through the portal, and these drive cyclic changes in the length of dsDNA as the phosphate groups respond to the protein's electrostatic potential. The DNA motions are captured by a coordinated protein-DNA grip-and-release cycle to produce DNA translocation. In short, the ATPase, portal, and dsDNA work synergistically to promote genome packaging.

Figure 1

The equilibrium conformation of DNA in the viral portals depends on the sequence and structure of the portal protein. These cross-sectional views show two of the protein chains from opposite sides of the dodecameric portal. Protein domains are colored using the scheme introduced by Sun et al. (39): green, blue, magenta, and orange identify the wing, stem, clip, and crown domains, respectively. The principal variations in DNA conformation (red) occur near the bottom of the alpha-helical stem region. Note that the P22 models include only the core region of the protein because the barrel domain is not resolved in the PC structure (4), and it was determined only at low resolution in the MV structure (40).

Figure 2

Protein-DNA contacts do not explain the DNA conformational changes. Gray, red, blue, and white spheres represent atoms of carbon, oxygen, nitrogen, and hydrogen, respectively, within 5 Å of any DNA atom for the four structures shown in Fig. 1. The scrunched region in ϕ29 has several contacts, particularly along one backbone, as described previously (35). In contrast, the stretched regions in the T4 and P22-PC DNAs have almost no direct contacts. Most important, the difference between the DNA conformations in P22-PC and P22-MV DNAs is clearly not due to direct contacts with the portal protein.

Figure 3

The DNA conformation responds to the electrostatic potential in the channel. The figures shows the electrostatic potential maps in the channels of the four portals in Fig. 1 (cross section). Positive (blue) and negative (red) contours are shown for ±0.25, ±0.5, ±1, ±2, and ±4 k_BT/e. In ϕ29, the phosphates are drawn toward a ring of positive charges lining the channel, causing the DNA to shorten. In T4 and P22-PC, the phosphates are repelled from regions of high negative potential, causing DNA lengthening. The electrostatic potential in the P22-MV channel is apparently not large enough to drive a significant conformational change in the DNA.

Figure 4

Model for DNA translocation driven by coupling cyclic changes in electrostatic potential with cyclic motions of two protein-DNA grips. (A) B-DNA (orange) in the channel of the complex of motor proteins (two protein chains shown schematically in green) is shown. The lower protein-DNA grip is closed at the bottom of the channel, and the upper grip is open (pairs of green triangles). (B) A conformational change in the proteins brings a negatively charged protein domain (pink) close to the DNA, driving the nearby DNA into an extended form (red). The head of the DNA is pushed forward into the capsid (arrow). (C) The upper grip closes to capture the DNA’s advance. (D) The lower grip opens. (E) A second conformational change in the protein pulls the negatively charged domain away from the DNA. The DNA returns to the B-form, pulling the tail of the DNA upward (arrow). (F) The lower grip closes to prevent DNA backsliding. (G) The upper grip opens, returning the protein to its original conformation. The DNA has advanced upward from its original position, which is marked by the dashed orange lines.