Structural framework for DNA translocation via the viral portal protein - PubMed (original) (raw)

Structural framework for DNA translocation via the viral portal protein

Andrey A Lebedev et al. EMBO J. 2007.

Abstract

Tailed bacteriophages and herpesviruses load their capsids with DNA through a tunnel formed by the portal protein assembly. Here we describe the X-ray structure of the bacteriophage SPP1 portal protein in its isolated 13-subunit form and the pseudoatomic structure of a 12-subunit assembly. The first defines the DNA-interacting segments (tunnel loops) that pack tightly against each other forming the most constricted part of the tunnel; the second shows that the functional dodecameric state must induce variability in the loop positions. Structural observations together with geometrical constraints dictate that in the portal-DNA complex, the loops form an undulating belt that fits and tightly embraces the helical DNA, suggesting that DNA translocation is accompanied by a 'mexican wave' of positional and conformational changes propagating sequentially along this belt.

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Figures

Figure 1

Figure 1

Bacteriophage SPP1 assembly. Double-stranded DNA is translocated into the procapsid through the portal protein, which together with the viral ATPase, forms a molecular motor. After termination of packaging, head completion proteins (gp15 and gp16) bind to the portal protein forming a head-to-tail connector. Tail attachment to the connector yields the infective phage particle.

Figure 2

Figure 2

X-ray structure of the SPP1 portal protein 13-subunit assembly. (A) Averaged, weighted 2∣_F_o∣−∣_F_c∣ electron density maps corresponding to the tunnel loops and viewed from the tunnel axis (vertical) toward the protein surface. The area with four tunnel loops (numbered 1–4) is outlined by dashed lines to show that there is room for mobility. One of the loops is fitted with a line to show its tilt with respect to the vertical axis. (B, C) Ribbon diagrams of the portal protein along and perpendicular to the 13-fold axis. (D) Single subunit with helices numbered to match the secondary structure of the φ29 portal protein (Simpson et al, 2000). The first and last residues of helices α3, α5 and α6 are indicated.

Figure 3

Figure 3

Location of mutations that affected specifically DNA packaging (Isidro et al, 2004a, 2004b). The SPP1 portal protein monomer is shown as a ribbon. The residues, shown as spheres centered at their Cα atoms, are subdivided into five groups: (red) residues in the tunnel loop and residues contributing to the negative charge of the tunnel's surface; (magenta) residues at the base of the portal protein that are likely to interact with the ATPase and gp15; (yellow) residues that could be involved in signal-force transmission between the portal protein and the ATPase and/or carry a structural role; (green) residues involved in stabilization of the crown; and (blue) residues with unassigned function.

Figure 4

Figure 4

Structural conservation. (A) Single subunits of SPP1 (cyan) and φ29 (yellow) portal proteins are superimposed. B-form DNA (van der Waals model) is positioned along the tunnel to show the relative size and match between the tunnel loop and the major groove of the DNA. (B) Secondary structure alignment of the central part of the polypeptide chain, α3–α6. For HK97, T4 and Epstein–Barr (EBV) portal proteins predicted secondary structures are shown. The positions of α3–α6 segments were validated by a number of criteria described in Materials and methods; the negatively charged residue at the tunnel entrance is highlighted in red. Tunnel loop sequences are shown as single letter code; the length of the cylinders (α-helices) and arrows (β-sheets) are proportional to the predicted length. (C, D) Molecular surfaces of two opposing subunits of SPP1 and φ29 portal proteins colored according to electrostatic potential. (E) Two extreme states of the SPP1 tunnel loop: the cyan from the crystal structure with residues stabilizing the kinked conformation of helix α6 shown in ball and stick, and the red obtained by modeling a straightened conformation of this helix.

Figure 5

Figure 5

Pseudoatomic structure of the 12-subunit assembly. (A–D) EM maps of the SPP1 connector (Orlova et al, 2003) with the fitted model of the portal protein. (A, B) Two orthogonal views of the connector. (C, D) Single subunit of the 12-mer (magenta), superimposed with a single subunit from the 13-mer (cyan). (E) Cα models of SPP1 (magenta) and φ29 (blue) portal proteins are fitted into five-fold averaged EM maps of φ29 proheads (Morais et al, 2005). The EM density is contoured at two different levels with the yellow map at a 25% lower contouring level than the pink map. For clarity, only a 16 Å slice of the maps and models is shown. (F) Changes in subunit–subunit contacts during 13- to 12-mer transition. Two diametrically opposite subunits of the 13-mer (left) and 12-mer (right) structures with van der Waals size of the tunnel are shown for the clip, tunnel loop and crown areas. Atoms forming short inter-subunit contacts (<3 Å) to the next subunit are highlighted in yellow. The two contact areas (dashed pink boxes) are different; the subunits in the 12-mer ‘roll' around the inter-subunit rotation axis (orange) to pack much more snugly. This forces substantial conformational changes in the crown and the tunnel loop (dashed blue ovals). The simple packing model schematized in black shows how the 12-mer tunnel diameter is considerably reduced by this rocking motion.

Figure 6

Figure 6

Energy landscape generated by the mismatching symmetries of portal protein and DNA. The _X_- and _Y_-axis correspond to the relative rotation and translation of the portal protein and the DNA, respectively. The relative scale on X and Y is such that distances on the drawing are proportional to those at the interface between the DNA and the tunnel loops. The energy minima form a periodic pattern. The horizontal separation between the minima corresponds to the observed 5.5 Å distance between the tips of the neighboring tunnel loops and the vertical separation corresponds to the 3.4 Å (1 bp) translation of the DNA. The combination of the two rotations (36–30°) effectively results in 6° horizontal angular separation between the minima, which corresponds to 1.1 Å horizontal displacement between the tips of the tunnel loops.

Figure 7

Figure 7

DNA translocation. (A, B) Top and side views of the pseudoatomic structure of the portal protein dodecamer with B-form DNA fitted into the tunnel. The double helix is shifted relative to the rotational axis of the portal protein (white cross/line) to avoid clashes with tunnel loops. (C) Stereo view of the proposed arrangement of tunnel loops (ribbons drawn along the main-chain atoms of residues 350–360) around the DNA (ball and stick). Loops occupying the three states inside the major groove are colored red, magenta and cyan, whereas the remaining nine loops are in dark blue. (D) Mechanistic model of DNA translocation. Two sequential states of the portal protein/DNA complex before (top) and after (bottom) consumption of one ATP molecule by the ATPase. A three-dimensional model (left) is sliced open (right) to give a representation with the horizontal axis corresponding to the angular position inside the tunnel; two periods of DNA are shown with two phosphates circled to provide reference points. The tilted tunnel loops (cylinders) and DNA (pink spheres centered at phosphates) are shown to scale. Numbers designate specific loops and colors designate specific conformational states. The three states—red, magenta and cyan—propagate along the circle of loops. There will be larger angular separation for the three loops occupying these three states, compared to the rest, allowing the three loops to dip into the major groove. An idealized mechanistic model of the transition between the top and bottom states requires 12° rotation of the bulk of the portal protein relative to the DNA. During this transition, loops 2–12 move with respect to the DNA, whereas loop 1 does not change its position with respect to the DNA, and together with the DNA moves by 6.8 Å relative to the capsid.

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