Conformational plasticity of RepB, the replication initiator protein of promiscuous streptococcal plasmid pMV158

Abstract

DNA replication initiation is a vital and tightly regulated step in all replicons and requires an initiator factor that specifically recognizes the DNA replication origin and starts replication. RepB from the promiscuous streptococcal plasmid pMV158 is a hexameric ring protein evolutionary related to viral initiators. Here we explore the conformational plasticity of the RepB hexamer by i) SAXS, ii) sedimentation experiments, iii) molecular simulations and iv) X-ray crystallography. Combining these techniques, we derive an estimate of the conformational ensemble in solution showing that the C-terminal oligomerisation domains of the protein form a rigid cylindrical scaffold to which the N-terminal DNA-binding/catalytic domains are attached as highly flexible appendages, featuring multiple orientations. In addition, we show that the hinge region connecting both domains plays a pivotal role in the observed plasticity. Sequence comparisons and a literature survey show that this hinge region could exists in other initiators, suggesting that it is a common, crucial structural element for DNA binding and manipulation.

Figure 1. OBD conformations in the C2 and C3 crystal structures of RepB.

(A) Views on the OBDs of the cartoon representations of the respective C2 and C3 crystal structures and after superposition using the ODs of both (C2 structure shown as a transparent surface in the superposition). The C2 protomer that is hexamerised to give the C6B model is indicated. The scale bars indicate the approximate dimension. (B) Views on the ODs of the cartoon representation of the C2 and C3 structures along the C6 axis of the ODs. The protomers are now alternately coloured red and blue, the dashed and continuous red outlines mark the boundaries of the OBD and OD, respectively, of one of the protomers. The figure shows that the OD of a blue-coloured neighbouring protomer is partly located above the OBD of a red-coloured protomer.

Figure 2. Superposition of the ODs of all nine protomers of the C2 and C3 structures showing the orientation of the OBDs with respect to the OD ring.

The OD hexamer is shown in blue and the different OBDs are represented by a ribbon, except for helix α2, which is drawn as a cartoon for visual reference. The OBDs are coloured from purple to red according to the distance to the central six-fold axis, where the OBD coloured purple is closest to that axis. (A) and (B) provide a lateral and a top view of the hexamer respectively, whereas (C) shows a view along the rotation axis relating the orientations of the OBDs.

Figure 3. Experimental SAXS curve of free RepB in solution (black dots) and calculated fits (solid lines).

(A) Fits from the individual atomic models C2, C3 and C6B are shown in green, blue and red, respectively. (B) Fits by the best mixtures of conformations: C2, C3 and C6B mix is shown in blue and the four representative MD snapshots (from panel C) in red. The inset emphasizes the differences in the fits in the region of the first shoulder of the SAXS profile. (C) Subset of four MD snapshots selected by EOM, which give the best fit according to OLIGOMER. The approximate symmetry of the models is indicated.

Figure 4. Average structures obtained from the last 10 nanoseconds of 100 nanoseconds long MD started from the C2 (3DKX), C3 (3DKY) and C6B (model) structures.

Figure 5. The control elements governing OBD movement in the RepB X-ray structures.

(A) The location of R130 at the OD/OBD interface and of the D and E residues of the N-terminal end of helix α5 that interact with R130 is shown. The insets show the position of the protomers in the C2 hexamer. (B) Views on the region where OBD conformation-dependent metal binding occurs at the different OD/OBDs interfaces of the structure cocrystallised with BaCl₂. The protein chain is represented by a cartoon drawing in all panels, except for the interdomain loop region (residues 128–134) containing the 3₁₀ helix, for which only a stick representation of the protein backbone is shown. The non-interacting side chains are left out for clarity. The neighbouring protomer is coloured blue and the Ba²⁺ ions are represented by a pink sphere. The contours represent the Ba²⁺ anomalous map (see text) contoured at 5σ. Direct and indirect contacts between protein residues and the metal ion, when present, are indicated by dashed lines. The OBD and OD are from two adjacent protomers and are colour-coded as in panels (A).

Figure 6. Predicted and observed secondary structure of the potential or defined domains of the pMV158 and viral Rep proteins discussed in the main text.

The amino-terminal end (N) and the number of amino acids are indicated for each protein analysed. The top horizontal line shows the result of the secondary structure prediction (predicted α-helices and β-strands are respectively represented by red and brown bars). The bottom horizontal line shows the observed secondary structure for the protein fragments whose structure is available (α-helices, β-strands and 3₁₀-helices are respectively represented by red, brown and blue bars). Conserved amino acid residues of the active site involved in metal binding (HUH) and in the endonucleolytic activity are indicated in the protein maps. The limits of the OBD, OD (either confirmed (OD) or potential (pOD) domain) and of the helicase domain are indicated in the protein maps. For RepB, AAV2 Rep, BPV E1 and SV40 LTag, the observed secondary structure of the ODs from the crystal structures are shown below the predicted secondary structure, preceded by the PDB entry code. Since there are no OBD structures available for the AAV2 and TGMV Reps, the C-terminal boundary of the OBD is shown as defined by alignment with the PF08724 and PF00799 Pfam families, respectively. Helicase domains of FBNYV and PCV2 Reps are defined by alignment with Pfam family PF00910 of SF3 helicases. The sequence and coordinates of the OBD-OD interdomain region that constitutes a potential hinge is indicated for every protein. The flexibility of each residue of the hinge predicted by the PredyFlexy server is represented by the gradient of colours indicated in the upper right corner of the figure. Grey colour represents a rigid residue whereas green and dark green colours represent residues with intermediate or high flexibility, respectively. The confidence index for the flexibility prediction is indicated for each residue just below the sequence. This index reflects the accuracy of the prediction and is represented by discrete values ranked from 1 to 19, with the prediction confidence increasing.

Figure 7. Comparison between the RepB structure and the structures of the viral initiators.

(A–C) Stereo views of the superposition of the ODs of BPV E1 helicase (PDB entry 2GXA, residues 308–378, light blue), AAV2 Rep (1S9H, residues 223–280, green) and SV40 LTag (1SVM, residues 266–355, yellow) on the RepB C2 structure (3DKX, residues 132–203, magenta). Superpositions were calculated using the SSM algorithm. For BPV E1 and SV40 LTag, the OBD-OD random-coiled hinge region is indicated. (D) Stereo view of a cartoon representation of the superposition of chain B of the C2 RepB hexamer on one of the OBD monomers of the AAV5 Rep in complex with its origin. AAV5 OBDs are coloured orange, the AAV5 DNA green, the RepB OBDs dark blue and the RepB ODs light blue.