Activation mechanism of a noncanonical RNA-dependent RNA polymerase

Garriga et al. 10.1073/pnas.0704447104.

Supporting Information

Files in this Data Supplement:

SI Table 1
SI Figure 5
SI Table 2
SI Figure 6
SI Figure 7
SI Figure 8




SI Figure 5

Fig. 5. Multiple sequence alignment of birnavirus RDRPs. The region shown only includes the sequence of the expressed polypeptide hVP1Δ846-879. IBDV (accession number Q8B540), Blotched Snakehead virus (BSNV, Q8AZL8), infectious pancreatic necrosis virus (IPNV, P22173), and Drosophila X virus (DrXV, Q91CD5). The strictly conserved residues are shown as red blocks, and similar residues in blue boxes. Secondary structure definitions for IBDV are drawn above the sequence alignment and colored by subdomain as in Fig. 1a. The conserved sequence motifs are marked by bars below the sequences and colored as in Fig. 2. Residues 251-263 of fingers form the motif G (pink). It is included within the loop connecting strand β6 and helix η4 and has a consensus sequence T/SX1-2GP. Strand β9 (residues 332-338; blue) contains motif F, which is unique to RDRP class and is defined as Arg-Xn-Ile/Leu (n=1, 2). Residues 396-406 of the VP1 palm contain motif C (yellow) that in Birnaviridae genera is present immediately upstream of motif A (residues 414-424; red). Motif C includes the AlaAspAsn sequence (residues 401-403) which is structurally similar to the canonical GlyAspAsp or SerAspAsp sequences found in cannonical RDRPs of know structure. Motif A includes the conserved residues Asp416 and Glu421. These acidic residues together with Asp402 seem to be the catalytic residues of Birnavirus RsRPs. Motif B (residues 490-500) that includes most of helix α13 is shown in green. The helix a14, together with the strand β15, forms the structural motif D (residues 515-524; purple). This motif provides the structural support to the central β-sheet containing motifs A and C. Motif E (residues 567-576; orange) is defined within the long loop joining the helix a15 and the strand β16 at the junction between the palm and the thumb subdomains.





SI Figure 6

Fig. 6. Electron density maps around the IBDV VP1 active site in the structure of the complex VP1-Mg2+. Stereoview of the final sA-weighted 2Fo-Fc Fourier map, contoured at 1.5 s, in the VP1 structure with the model placed inside (ball and sticks colored in atom type code). Three Mg2+ ions were found in the structure, two of them in the polymerase active site, bound to the acidic residues Asp402 (motif C) and, Asp416 and Glu421 (motif A) in a position close to the expected for the catalytic metals. The third metal ion is bound at a site ~6Ã… from the expected catalytic positions. The octahedral coordination in this site constructed from the carboxylate oxygens of Asp-416 and Asp-531, the carbonyl oxygen of Ile-415, the hydroxyl groups of Asn-403, Tyr-406, and Ser-414, and one water molecule. The Mg2+ ions, located close to acidic residues are shown as orange balls. Water molecules are shown as green balls.





SI Figure 7

Fig. 7. Conformational changes of the B loop induced by VP3 peptide binding. (a) Superimposition of the secondary structural elements, containing motif B of the unbound (green) and VP3 peptide bound (yellow) structures of the polymerase VP1. (b) Stereoview of the final σA-weighted 2Fo - FcFourier map (1.5σ) that shows the B loop region in the unbound VP1 structure. (c) σA-weighted 2Fo - FcFourier map (1.2σ) of the equivalent region in the VP1-VP3 complex.





SI Figure 8

Fig. 8. Structure-based sequence alignment of the polymerase motifs (A, B, C, E, F and G) of the RDRPs from IBDV, FMDV and Φ6. The strictly conserved residues are shown as red blocks, and similar residues in blue boxes. The amino acids that are predicted to be in contact, either with the template, primer, or the incoming NTP, are marked by arrowheads. The structural motif D does not show sequence conservation.