Revolution rather than rotation of AAA+ hexameric phi29 nanomotor for viral dsDNA packaging without coiling

Abstract

It has long been believed that the DNA-packaging motor of dsDNA viruses utilizes a rotation mechanism. Here we report a revolution rather than rotation mechanism for the bacteriophage phi29 DNA packaging motor. The phi29 motor contains six copies of the ATPase (Schwartz et al., this issue); ATP binding to one ATPase subunit stimulates the ATPase to adopt a conformation with a high affinity for dsDNA. ATP hydrolysis induces a new conformation with a lower affinity, thus transferring the dsDNA to an adjacent subunit by a power stroke. DNA revolves unidirectionally along the hexameric channel wall of the ATPase, but neither the dsDNA nor the ATPase itself rotates along its own axis. One ATP is hydrolyzed in each transitional step, and six ATPs are consumed for one helical turn of 360°. Transition of the same dsDNA chain along the channel wall, but at a location 60° different from the last contact, urges dsDNA to move forward 1.75 base pairs each step (10.5bp per turn/6ATP=1.75bp per ATP). Each connector subunit tilts with a left-handed orientation at a 30° angle in relation to its vertical axis that runs anti-parallel to the right-handed dsDNA helix, facilitating the one-way traffic of dsDNA. The connector channel has been shown to cause four steps of transition due to four positively charged lysine rings that make direct contact with the negatively charged DNA phosphate backbone. Translocation of dsDNA into the procapsid by revolution avoids the difficulties during rotation that are associated with DNA supercoiling. Since the revolution mechanism can apply to any stoichiometry, this motor mechanism might reconcile the stoichiometry discrepancy in many phage systems where the ATPase has been found as a tetramer, hexamer, or nonamer.

Fig. 1

Depiction of structure and function of phi29 DNA-packaging motor. (A) Illustrated model of hexameric pRNA based on a crystal structure (Zhang et al., accepted for publication) and the 30° tilting of the channel subunits, relative to the central axis of the connector (pdb ID: 1H5W); (B) DsDNA showing the shift of 30° angle between two adjacent connector subunits; (C) Connector showing the change of 30° angle between two adjacent connector subunits; (D) AFM images of hexameric pRNA with 7-nt loops (Shu et al., 2013).

Fig. 2

FRET assay of fluorogenic ATPase and short dsDNA. eGFP-gp16 was incubated with Cy3-DNA and without ATP and excited at 480 nm. Energy transfer occurs between the two fluorophores, with light emission at ~560 nm, indicating that gp16 and DNA are in close proximity. Bar graph (top right) showing the FRET efficiency difference between the two samples.

Fig. 3

EMSA of eGFP-gp16 on terminally blocked short dsDNA. 40 bp Cy3-dsDNA, with biotin attached to each end, was incubated with eGFP-gp16, non-hydrolyzable γ-S-ATP, and streptavidin in different combinations. The complexes that were mixed at approximately a 6:1 molar ratio of protein:DNA were then electrophoresed through an agarose gel and scanned for Cy3 fluorescence of DNA and GFP fluorescence of gp16 (see Materials and Methods).

Fig. 4

Binomial distribution assay to determine the minimum number (y) of defective eGFP-gp16 in the hexameric ring to block motor activity. Theoretical plot of percent Walker B mutant gp16 versus yield of infectious virions in in vitro phage assembly assays. Predictions were made with the equation as seen in the Materials and Methods.

Fig. 5

One γ-S-ATP is sufficient to bind to one subunit of the gp16 hexamer and promote a high affinity state for dsDNA. Sequential binding of gp16 for dsDNA substrate involves γ-S-ATP substep. (A) The K_d for dsDNA at varying concentrations of γ-S-ATP. (B) The relative K_d of gp16 decreased 40-fold as the concentration of γ-S-ATP increased from 0 mM to 1 mM. (C) ADP, a derivative of ATP hydrolysis, was unable to promote binding and had the similar effect as no nucleotide addition. The hyperbolic curve (D) suggests a cooperativity factor of 1, indicating that one γ-S-ATP is sufficient to produce the high affinity state of gp16 for DNA. DNA releases from the complex DNA-gp16-γ-S-ATP mediated by ADP (E), forming a sigmoidal curve (F) with a cooperativity factor of 6 indicating that all six subunits of gp16 need to be bound to ADP to release DNA from the protein.

Fig. 6

ATPase inhibition assay by Walker B mutants reveals complete negative cooperativity. The inhibition ability of the Walker B mutants E119A and D118E/E119D was assayed by ATPase activity in the absence (left) and presence (right) of dsDNA. In the presence of DNA (right), the experimental data (solid line) overlapped with a theoretical curve indicating that one inactive subunit (dotted line) within the hexamer is able to completely block the activity of the hexameric gp16 and abolish ATPase activity, demonstrating negative cooperativity (see also Fig. 4). The dashed line is the theoretical curve where two inactive subunits are necessary for inhibition.

Fig. 7

Schematic of gp16 binding to DNA and mechanism of sequential revolution in translocating genomic DNA. The connector is a one way valve that allows dsDNA to move into the procapsid, but does not allow movement in the opposite direction. Gp16, which is bridged by pRNA to associate with the connector, provides the pushing force. The binding of ATP to one subunit stimulates gp16 to adopt a conformation with a higher affinity for dsDNA. ATP hydrolysis forces gp16 to assume a new conformation with a lower affinity for dsDNA, thus pushing dsDNA away from the subunit and transferring it to an adjacent subunit. DsDNA is translocated at a pace of 1.75 base pairs per transfer to the neighboring subunit and is bound at a location 60° different from the first subunit on the same phosphate backbone chain. Rotation of neither the hexameric ring nor the dsDNA is required since the dsDNA revolves around the diameter of the ATPase. In each transitional step, one ATP is hydrolyzed, and in one cycle, six ATPs are required to translocate dsDNA one helical turn of 360° (10.5 base pairs). An animation is available at http://nanobio.uky.edu/movie.html.

Fig. 8

Part I. Direct observation of ATPase complex queued and moving along dsDNA. Cy3 conjugated gp16 was incubated with (A, B, E) and without (D) dsDNA, tethered between two polylysine beads where (C, F) are magnified images of the framed regions of (B, E), respectively. (A–C) are overlapped pseudocolor images indicating the binding of Cy3-labeled gp16 along the To-Pro-3 stained dsDNA chain (Red: Cy3-gp16; Green: To-Pro-3 DNA). (G, H) The motion of the Cy3-gp16 spot was analyzed and a kymograph was produced to characterize the ATPase walking. (Actual motion videos can be found in the supplementary information and at http://nanobio.uky.edu/movie.html). Part II. Negatively stained transmission electron microscopy images of ATPase queued along dsDNA. gp16 was bound to non-specific dsDNA in queue. Part III. Recording of two Cy3-gp16/dsDNA complexes showing motionless gp16 spots in a buffer containing no ATP. (A) Sequential images of the recording. (B) Kymograph of the two spots.

Fig. 9

Binding affinity of gp16 to dsDNA and procapsid/pRNA complex measured using sucrose sedimentation. Ratio of procapsid-bound and DNA-bound gp16 under different treatments where the percent of bound gp16 to total gp16 is expressed, showing gp16’s affinity to DNA is much greater than to procapsid/pRNA complex.

Fig. 10

Mechanism of sequential revolution in translocating genomic DNA. Connector is a one way valve (Jing et. al., 2010) that allows dsDNA to move into the procapsid, but does not allow movement in the opposite direction. (A) Binding of ATP to one gp16 subunit stimulates it to adapt a conformation with higher affinity for dsDNA. ATP hydrolysis forces gp16 to assume a new conformation with lower affinity for dsDNA, thus pushing dsDNA away from this subunit and transferring it to an adjacent subunit. (B) Binding of gp16 to the same phosphate backbone chain, but at a location 60° different from last subunit urges dsDNA to move forward 1.75 base pairs. Since the dsDNA chain is transferred from one point on the phosphate backbone to another point, the rotation of the hexameric ring or the dsDNA is not required. (C) The revolution of dsDNA along the 12 subunits of the connector channel.

Fig. 11

DNA revolves and transports through 30° tilted connector subunits facilitated by anti-parallel helices between dsDNA helix and connector protein subunits. The anti-parallel configuration can be visualized in an external view (A) in which DNA revolves through the connector making contacts at every 30° subunit (B,C). A planar view is suggested (D) in which DNA is advanced and travels along the circular wall of the connector channel with no torsion or coiling force, through the connector channel, touching each subunit translating to 12 discrete steps of 30° revolving turns for each step.