Research ArticleSTRUCTURAL BIOLOGY

A viral genome packaging motor transitions between cyclic and helical symmetry to translocate dsDNA

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Science Advances  07 May 2021:
Vol. 7, no. 19, eabc1955
DOI: 10.1126/sciadv.abc1955
  • Fig. 1 The bacteriophage phi29 dsDNA packaging motor.

    (A) Cutaway side view of the bacteriophage phi29 dsDNA packaging motor as determined by cryo-EM. Molecular envelopes of the connector, pRNA, and ATPase are shown in cyan, magenta, and blue, respectively. (B) Model of the mechanochemical cycle of the packaging motor as determined by single-molecule experiments [adapted from Chistol et al., 2012 (32)]. The top and bottom halves of the panel show the chemical and mechanical transitions in the dwell phase and burst phases, respectively. The presumed mechanochemical state of the motor trapped by ATP-γ-S is indicated by an asterisk.

  • Fig. 2 Reconstruction of particles stalled during packaging.

    (A) Side view of stalled-particle reconstruction, colored by cylindrical radius. Components of the packaging motor are colored magenta. (B) Zoom in on one capsid hexamer, outlined in red in (A). (C) The top panel shows the atomic model of the capsid protein built into density corresponding to one monomer in the hexamer shown in (B). The bottom panel shows the ribbon diagram with the N-terminal HK97 and the C-terminal immunoglobulin (IG)–like domains colored blue and pink, respectively. (D) Cross-section of the reconstruction of stalled particles colored as in (A) but with packaged DNA colored red. (E) Focused C12 reconstruction of the portal. The top panel shows an end-on view with the fitted portal structure (yellow ribbon; PDB ID: 1FOU). The lower panel shows a side view of the central helical domain. (F) Focused C1 reconstruction of the entire portal vertex with the atomic model of the portal built into its corresponding density. The top and middle panels show end-on views looking from inside the capsid (upper) and from below the portal (middle); the portal, pRNA, and capsid are shown in yellow, magenta, and blue, respectively. The bottom panel shows the side view. (G) The top panel shows the structure of a fragment of the pRNA (PDB ID: 4KZ2) fitted into its corresponding density. The bottom panel shows the atomic resolution structure of the pRNA built into its corresponding density. The E loop of the pRNA attaches to E loops in the capsid protein, colored blue, green, and red.

  • Fig. 3 Cryo-EM reconstruction of the phi29 DNA packaging ATPase motor.

    (A) Density for the CTD planar ring viewed looking from below (left) and above (right). Five copies of the CTD NMR structure (PDB ID: 6V1W) are fitted into their corresponding densities and shown as red, yellow, green, orange, and blue ribbon diagrams. (B) Density for the NTD helical assembly viewed looking from below (left) and above (right). Five copies of the NTD crystal structure (PDB ID: 5HD9) are fitted into their corresponding densities and shown as red, yellow, green, orange, and blue ribbon diagrams. (C) A cutaway side view of the phi29 ATPase motor, with CTD and NTD ribbon diagrams colored as above. The pRNA (periphery) and DNA (center) are shown as tan ribbon diagrams in all three panels.

  • Fig. 4 Structure of the bacteriophage phi29 dsDNA packaging motor stalled during packaging.

    Structure of the packaging motor rendered as molecular surfaces (top panels) and ribbon diagrams (bottom panels) shown from the following: (A) side view; (B) cutaway side view to visualize the DNA in the central channel; and (C) an end-on view, looking from below the motor. The portal and pRNA are colored cyan and magenta, respectively, and the five ATPase subunits are labeled S1 to S5 and colored blue (S1), orange (S2), green (S3), yellow (S4), and pink (S5). Approximate levels of the CTD and NTD are indicated by blue arrows in the panel. Deviations from the rotational component of helical symmetry are shown indicating loose and tight interfaces by red and green asterisks, respectively, in (C); the loose interfaces are on either side of the lowest subunit in the ATPase helix and correspond to the two active sites where there is no clear density for ATP. Two black lines in the bottom half of (B) are drawn approximately coincident with the helical axis of the DNA before and after the kink that occurs between the CTD and NTD; the ~12.5° angle between the lines is indicated.

  • Fig. 5 Helical arrangement and offset of ATPase NTDs.

    (A) Side view of the ATPase NTDs showing their helical arrangement. The NTDs from five different subunits are labeled S1 to S5 and colored as in Fig. 4. ATP is colored by element; (B) K56 is arranged as a spiral that approximately tracks the DNA helix. The bottom four subunits track the 5′-3′ strand approximately every 2 bp. Because of the imperfect NTD helical symmetry, the top K56 is closer to the complementary 3′-5′ strand. (C) Helical symmetry axes for successive superpositions of S1 on remaining subunits are shown as colored rods and labeled along with their actual and ideal (parentheses) rotations. Translational components of the helical operations are represented by the offsets of the rods. (D) COM of the NTD and CTD are shown as gray spheres from side and end-on views. The fivefold axis of the phage is shown as a darker gray rod. (E) S1 (blue) and its associated pRNA (magenta ribbon and translucent surface). The approximate portal-binding surface and the unique NTD-pRNA contact in S1 are shown as cyan and blue stars, respectively. (F) Superpositions of the pRNA from side and end-on views. The pRNAs were superimposed onto pRNA-S1 via their prohead-binding domains (bases 1 to 74) and colored according to their associated NTDs. The blue pRNA-S1 is oriented as in (E); note that S1 and S2 pRNAs are bent relative to the others such that its distal NTD-binding regions move up and to the right.

  • Fig. 6 Domain linker and trans-acting residues.

    (A) Side view and (B) end-on view of adjacent subunits S1 and S2, colored as in Figs. 6 and 7. NTDs and CTDs are labeled, and the linker domain in S2 is highlighted in yellow. ATP is shown as space-filling spheres colored by element. (C) Superposition of the NTDs of all five subunits to illustrate structural variability of the linker domain. Note that the relative orientation of linker domains from subunits S1 (blue) and S5 (pink) differ the most and correspond to subunits at the top (S1) and bottom (S5) of the ATPase helix. (D) Close up of the active site between subunits S1 and S2. Residues important for binding and/or catalysis, including K105 and R146, are shown as ball and stick figures, colored by heteroatom, and labeled. ATP atoms are shown as ball and stick and colored by element; note that phosphorous is colored light purple rather than the typical orange to facilitate visibility.

  • Fig. 7 Mechanism of DNA translocation.

    For simplicity, only the NTDs of the ATPase are shown, and subunit positions have been adjusted to obey perfect helical symmetry as described in the text. NTDs from the five different subunits, labeled S1 to S5, are colored differently and shown as semitranslucent surfaces. ATP and PO4 are rendered as opaque molecular surfaces and colored by element. DNA is shown in blue, with the B-form DNA helical repeat indicated by coloring every 10 bp on the 5′-3′ strand orange. The top panel shows the burst phase; ATP hydrolysis causes the NTDs to transition from a helical to a planar configuration and drive DNA into the procapsid (top of page). Note that while the first four hydrolysis events move 10 bp of DNA in four 2.5-bp steps, the last hydrolysis event in S5 does not translocate DNA. The bottom panel illustrates the dwell when sequential exchange of ADP for ATP occurs, and the motor resets to the helical configuration as NTD subunits S2 to S5 walk down DNA. Note that during the dwell, there is no translocation of DNA.

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