Volume 157, Issue 3, Pages 702-713 (April 2014) A Viral Packaging Motor Varies Its DNA Rotation and Step Size to Preserve Subunit Coordination as the Capsid Fills  Shixin Liu, Gheorghe Chistol, Craig L. Hetherington, Sara Tafoya, K. Aathavan, Joerg Schnitzbauer, Shelley Grimes, Paul J. Jardine, Carlos Bustamante  Cell  Volume 157, Issue 3, Pages 702-713 (April 2014) DOI: 10.1016/j.cell.2014.02.034 Copyright © 2014 Elsevier Inc. Terms and Conditions

Cell 2014 157, 702-713DOI: (10.1016/j.cell.2014.02.034) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Overview of the φ29 Packaging Motor (A) Cryoelectron microscopy reconstruction of the φ29 capsid (gray), connector (cyan), pRNA (magenta), and gp16 ATPase (blue). Reproduced from Morais et al. (2008), with permission from Elsevier. (B) Mechanochemical model of the dwell-burst packaging cycle at low capsid filling (Chemla et al., 2005; Chistol et al., 2012; Moffitt et al., 2009). Cell 2014 157, 702-713DOI: (10.1016/j.cell.2014.02.034) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 The φ29 Motor Rotates DNA during Packaging (A) Left: experimental geometry of the rotation assay. The packaging complex is tethered between two beads. Biotin-streptavidin linkages torsionally couple the rotor bead to the optically trapped bead via dsDNA. A nick and a ssDNA region ensure that the rotor bead is torsionally decoupled from the micropipette-bound bead. Middle view is a micrograph of the experimental geometry. Right view is a kymograph of the rotor bead position during packaging, displayed at 5 Hz. (B) Sample traces displaying DNA tether length (top), rotor bead angle (middle), and torque stored in the DNA (bottom) during packaging. Pauses in translocation are concomitant with pauses in rotation (shaded light red). Slipping (shaded light blue) causes a reversal in the rotation direction. (C) Sample packaging traces with intact proheads (purple) and trepanated proheads (magenta). (D) Local DNA rotation density (ρ) versus capsid filling. The data point obtained with trepanated proheads—corresponding to very low capsid filling conditions—is shown as a magenta square. Error bars represent SEM. (E) The geometric basis for DNA rotation at low capsid filling. Left view is a B-form DNA backbone diagram (only the 5′–3′ strand is shown). The φ29 motor forms specific contacts with pairs of phosphates (red) every ten bases. Right is a diagram of the motor (blue) and the DNA (orange) as viewed from within the capsid. The same subunit contacts the DNA backbone phosphates in consecutive dwells. After a 10 bp burst, a 14° clockwise DNA rotation is needed to bring the DNA and the motor into perfect register. See also Figure S1 and Movie S1. Cell 2014 157, 702-713DOI: (10.1016/j.cell.2014.02.034) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Motor Coordination Is Preserved at High Capsid Filling (A) Experimental geometry of the high-resolution packaging assay. (B) Sample traces displaying individual packaging cycles at various levels of capsid filling. Raw 2,500 Hz data are shown in gray, and downsampled 100 Hz data are in black. Stepwise fit to the data highlights dwells and bursts in red and green, respectively. (C) Cumulative probability distribution of the packaging cycle times. Each color corresponds to a certain filling level. (D) The nucleotide exchange scheme for each ATPase subunit. (E) Sample packaging traces at low external loads (7–10 pN) and various ATP concentrations. (F) Mean packaging velocity (pauses and slips removed) versus [ATP]. Each color represents a different filling level. The data are fit to the Hill equation: v = Vmax × [ATP]n/(KMn + [ATP]n). Inset shows the Hill coefficient n from the fit. (G) Inverse of the mean packaging velocity versus the ratio of [ADP] to [ATP] at different filling levels. [ATP] is fixed at 250 μM. The data are fit to a competitive-inhibition model, 1v=1Vmax(1+KM[ATP]+KMKI[ADP][ATP]). Inset shows the dissociation constant for ADP, KI, from the fit. In (F) and (G), error bars represent 95% confidence intervals (CI) estimated via bootstrapping. See also Figure S2. Cell 2014 157, 702-713DOI: (10.1016/j.cell.2014.02.034) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 High Capsid Filling Slows ATP Tight Binding and Induces LLPs (A) The chemical cycle of each φ29 ATPase subunit. (B) Vmax (blue) and KM (green) versus capsid filling. (C) Vmax/KM versus capsid filling. (D) Mean dwell duration versus capsid filling at saturating [ATP]. (E) Apparent number of rate-limiting kinetic events during the dwell versus capsid filling. (F) Sample packaging traces at various filling levels highlighting regular dwells and LLPs. (G) Sample traces highlighting the locations of LLPs. For clarity, only LLPs longer than 5 s are shown. LLPs from different packaging complexes are colored green, blue, black, and red. (H) Mean LLP duration versus capsid filling at saturating [ATP]. (I) Frequency of LLP occurrence versus capsid filling at saturating [ATP]. Error bars represent 95% CI. See also Figures S3 and S4. Cell 2014 157, 702-713DOI: (10.1016/j.cell.2014.02.034) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 Capsid Filling Modulates the Burst Duration (A) Sample packaging traces collected at low capsid filling (15%–30%) and various external loads. Raw 2,500 Hz data are shown in gray, and downsampled 100 Hz data are in black. Stepwise fit to the data highlights dwells and bursts in red and green, respectively. (B) Mean burst duration versus external force at low capsid filling (15%–30%). The data are fit to an Arrhenius-type equation: τburst(F) = τburst(0) × eFΔx/kT (dashed curve, Δx = 0.33 ± 0.08 nm from the fit). Inset shows mean dwell duration versus external force. (C) Mean burst duration versus capsid filling. (D) The magnitude of the internal force as a function of capsid filling, obtained by applying the τburst-F curve (B) to the τburst-filling dependence (C). Error bars represent 95% CI. Cell 2014 157, 702-713DOI: (10.1016/j.cell.2014.02.034) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Capsid Filling Modulates the Step Size of the Motor without Affecting Subunit Coordination (A) Mean burst size versus capsid filling. (B) Sample packaging traces at high capsid filling and high external loads revealing fragmented bursts. Raw 2,500 Hz data are shown in gray, downsampled 150 Hz data in black, and stepwise fits in blue. (C) Histogram of burst fragment sizes at high force and high filling. The distribution is well fit by two Gaussians that correspond to individual 2.3 bp steps and 4.6 bp burst fragments consisting of two consecutive 2.3 bp steps. (D) The ρ values inferred from the observed burst sizes (gray) and the measured ρ values (purple and magenta) as a function of capsid filling. (E) Illustration of how a smaller burst size results in a larger amount of DNA rotation (compare to Figure 2E). Error bars represent 95% CI. See also Figure S5. Cell 2014 157, 702-713DOI: (10.1016/j.cell.2014.02.034) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 The φ29 Motor Adjusts Its Operation in Response to Increasing Capsid Filling (A) Average time needed to package 100 bp of DNA at different filling levels. Each color denotes a distinct mechanism that contributes to the slowing down of the motor as the capsid fills. (B) Characteristics of packaging at low capsid filling and high capsid filling. The φ29 packaging cycle consists of a dwell phase (red) during which five ATPs are loaded sequentially, and a burst phase (green) during which four subunits translocate DNA. In each cycle, the motor rotates the DNA to form specific electrostatic contacts with the DNA backbone. These contacts anchor the motor onto the DNA during the subsequent dwell and determine the identity of the special subunit. Although rotation is depicted here to occur at the end of the burst, alternative scenarios where rotation occurs elsewhere during the cycle cannot be strictly ruled out. Several mechanisms modulate the motor operation at high filling while preserving the overall motor coordination: (1) the ATP tight-binding rate is downregulated, prolonging the dwell; (2) the motor enters the LLP state; (3) the duration of the force-sensitive burst phase is prolonged, most likely due to the rising internal force; (4) the step size of the motor decreases, which results in smaller bursts; and (5) the amount of DNA rotation per cycle increases. D, ADP-bound subunit; T, ATP-bound subunit. Cell 2014 157, 702-713DOI: (10.1016/j.cell.2014.02.034) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S1 Corrections, Controls, and Models for the DNA Rotation Experiment, Related to Figure 2 (A) Correcting rotor bead motion for hydrodynamic drag to calculate DNA rotation by the φ29 packaging motor. The rotation angle for the two sample traces shown in Figure 2B is plotted as the observed rotor bead motion (blue) and the derived DNA rotation (cyan). See Extended Experimental Procedures for the detailed procedure of deriving DNA rotation. (B) Analysis of the simulated bead-rotation signal. In the simulation, the mock DNA was rotated by the packaging motor at a filling-dependent rotation density (red line). The resulting motion of the rotor bead, accounting for biological, thermal, and instrumental noise and error, was simulated (Extended Experimental Procedures). The simulated motion was then analyzed using the same procedure as the experimental data shown in Figure 2D, illustrating that the original filling dependence of DNA rotation can be recovered (black circles; error bars are SEM), thus validating our approach. (C) Sequence independence of the rotation density. The DNA backbone twist across each base pair was computed for the sequences used in our experiments, based on previously reported values (Wynveen et al., 2008). The results were averaged over every 100-bp window. Note that there were two possible DNA substrates of nearly identical length that could be packaged in these experiments (see Extended Experimental Procedures for details). The local helical twists of the two possible substrates are identical at 34.6 degrees per base pair along the entire sequences (blue and green). If the observed change in rotation density were simply a sequence-dependent effect, the twist per base pair would be required to drop substantially toward the end of the sequence (red), contrary to our calculations. (D) Simultaneous DNA translocation and rotation by packaging motors on trepanated proheads. Sample packaging traces (dotted lines – complex 1; solid lines – complex 2) show that motors on trepanated proheads rotate DNA (blue) during translocation (red). Trepanated proheads were enriched by freeze-thaw cycling of pRNA-free proheads and subsequently adding pRNA onto the prohead. (E and F) Putative scenarios in which the DNA-contacting subunit precesses around the ring in successive cycles. (E) The special subunit that makes specific contacts with DNA backbone phosphates precesses around the pentameric ring counter-clockwise by one subunit after each packaging cycle, producing a +58° DNA rotation per cycle. (F) The DNA-contacting special subunit precesses clockwise by one subunit after each cycle, yielding a −86° DNA rotation per cycle. Neither scenario is compatible with the rotation density obtained experimentally (see Extended Experimental Procedures for further discussion). Cell 2014 157, 702-713DOI: (10.1016/j.cell.2014.02.034) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S2 Sample Packaging Traces at Various ADP Concentrations, Related to Figure 3 These experiments were performed at a fixed ATP concentration of 250 μM. The DNA packaging velocity at a given capsid filling decreases as the ADP concentration increases (Figure 3G). Cell 2014 157, 702-713DOI: (10.1016/j.cell.2014.02.034) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S3 Using Simulations to Explore the Effect of Each Kinetic Rate in the Chemical Cycle of the φ29 ATPase on the Michaelis-Menten Parameters, Related to Figure 4 (A–C) Dependence of the maximum velocity Vmax, the Michaelis-Menten constant KM, and their ratio Vmax/KM on the ATP-tight-binding rate (A), the ATP-docking rate (B), and the ATP-undocking rate (C). Vmax is sensitive to changes in kATP_tight_bind, but is not affected by changes in kATP_dock or kATP_undock. (D) Vmax/KM is insensitive to changes in the rate of ADP release, Pi release, ATP hydrolysis, or condensation of ADP and Pi. Cell 2014 157, 702-713DOI: (10.1016/j.cell.2014.02.034) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S4 Characterizing the Properties of LLPs, Related to Figure 4 (A and B) LLPs occur predominantly during the dwell phase. (A) Left: A sample packaging trace segment shows regular dwells and bursts (blue) and the beginning of an LLP (red). Raw 2500-Hz data are shown in gray. Downsampled 100-Hz data are shown in blue and red. Right: Residence time histogram computed from the downsampled data as previously described (Chistol et al., 2012). The largest peak (red arrowhead) corresponds to the location of the LLP and the smaller peaks correspond to the locations of regular dwells. Two regular dwells serve as anchors (blue arrowheads) for inferring the size of the burst immediately before the LLP and aligning different LLP-containing segments. To remove any filling-dependent variation in the burst size, the segments were scaled such that the separation between the two anchors is always 10 bp. (B) Residence time histograms (thin gray curves) from 159 LLP-containing segments were aligned using their anchor dwells (blue arrowheads at 0-bp and 10-bp) and averaged (thick blue curve). The averaged histogram shows that the LLP peak is located around −20 bp (red arrowhead), i.e., integer multiples of a full burst size from the anchor dwells, indicating that most of the LLPs occur during the dwell phase of the motor’s packaging cycle. Inset: Distribution of pre-LLP burst sizes shows a major peak at 10 bp (after the aforementioned scaling to remove the filling dependence of the burst size), again indicating that most LLPs occur during the dwell. (C and D) Distinguishing LLPs from regular dwells. Dwell time distributions (gray) at low (C) and high (D) capsid filling were fit by Gamma distributions (blue curves). Insets display the tails of the distributions. The long events unaccounted for by the Gamma distributions were designated as LLPs. At 15%−30% filling, the longest event is ∼0.5 s, whereas at 95%−100% filling, the longest event is ∼35 s. See Extended Experimental Procedures for further discussion. Cell 2014 157, 702-713DOI: (10.1016/j.cell.2014.02.034) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure S5 Sizes of the Bursts and Burst Fragments at Different Levels of Capsid Filling, Related to Figure 6 (A) Distributions of burst sizes at different levels of capsid filling. Each distribution can be fit by a sum of Gaussian distributions representing integer 10-bp, 9-bp, and 8-bp burst sizes (dashed black, blue, and red curves, respectively). Solid green curves represent the shapes of the summed distributions. Vertical dashed lines represent the mean burst sizes. (B) Two sample packaging traces at high capsid filling (80%−100%) and low external loads (7−10 pN) exhibiting burst fragmentation (black arrows). Raw 2500-Hz data are shown in gray and downsampled 100-Hz data in black. Stepwise fit to the data highlights dwells and bursts in red and green, respectively. Under these conditions, bursts are either instantaneous or broken up into two resolvable fragments. s denotes the size of the first segment and b denotes the size of the entire burst. (C) Normalized sizes of the first resolvable burst segments, s/b, at different levels of capsid filling. The mean value of s/b is ∼0.5 at all filling levels, consistent with four steps making up each burst and two steps making up each resolvable burst segment. Cell 2014 157, 702-713DOI: (10.1016/j.cell.2014.02.034) Copyright © 2014 Elsevier Inc. Terms and Conditions