Fig. 2. Topological diagram of the consensus fold of AdoMet-dependent MTases (Fauman et al., 1999), NS5MTase_DV and VP39 (Hodel et al., 1996). Triangles represent strands and circles represent helices. The positions of the N- and C-termini are represented by squares. The elements of the consensus AdoMet-dependent MTase fold are coloured in light grey. Those that are replaced by different elements (loops or β-strand) in NS5MTase_DV or VP39 are dashed. AdoMet or AdoHcy bound at the C-terminus of the β-sheet is shown in black. The shape and dimension indicate which secondary structural elements are included in its contact zone. N- or C-terminal appendages of the MTase core in both NS5MTase_DV and VP39 are shown in black and dark grey, respectively.

Fig. 7. The GTP-binding site of NS5MTase_DV. (A) Stereo view of the experimental (F_o – F_c) difference map (2.9 Å) contoured at 3σ in the vicinity of Phe25 in a GDPMP-soaked crystal. The C_α trace is shown in bold sticks and coloured as in Figure 1B and C. Side chains are shown in stick representation and coloured according to atom type. For clarity, non-interacting side-chains of residues 17, 19 and 20 are not shown. Dotted lines indicate hydrogen bonds between GDPMP and NS5MTase_DV. (B) Electrostatic surface representation of the MTase active site cleft and of the positively charged GDPMP binding site (framed area of Figure 3A). The figure was generated with GRASP (Nicholls et al., 1991) and coloured as in Figure 3A. (C) Mutational analysis of NS5MTase_DV. Purified NS5MTase_DV mutants (2 µg) were tested for their ability to bind [α-³²P]GTP, as described for wild-type NS5MTase_DV in the legend to Figure 6A.

Fig. 3. The AdoMet/AdoHcy-binding site. (A) General view of the position of AdoHcy within NS5MTase_DV. The solvent-accessible surface of NS5MTase_DV was calculated and is displayed using GRASP (Nicholls et al., 1991). It has been coloured according to electrostatic potential. Potential values range from –25 kT (red) to 0 (white) to +25kT (blue), where k is the Boltzman constant and T is the temperature. The AdoHcy molecule is shown in stick representation. The area indicated by the red dotted square delineates the sector shown in detail later in Figure 7B. (B) Stereo diagram of the AdoMet/AdoHcy-binding site in NS5MTase_DV. Carbons are displayed in yellow, oxygens in red, nitrogens in cyan and sulfur in green. The F_o – F_c difference map, contoured at 3σ, was calculated at 2.4 Å resolution from a model in which the ligand was omitted. It is clear from the electron density map that the molecule is a co-product of the methyltransfer, i.e. the AdoHcy molecule. Main hydrogen bonds between NS5MTase_DV and AdoHcy are indicated by black dotted lines. Also shown is a sulfate ion, originating from the crystallization conditions. This sulfate ion is hydrogen-bonded to the 2′- and 3′-oxygen of the AdoHcy ribose. For clarity, two water molecules and Tyr219, which are also interacting via hydrogen bonds, are not represented.

Fig. 4. MTase activity. (A) Assay of MTase activity. The extent of methyltransfer from Ado[methyl-³H]Met to three different RNA substrates (pppACCCCC, GpppACCCCC and ^7MeGpppACCCCC) by 5 µg of NS5MTase_DV is plotted as a function of time. Data points represent the averages of three independent experiments and are presented as percentage of [methyl-³H] incorporation. The plateau of 100% incorporation represents a concentration of 1.5 µM transferred methyl groups in the reaction at the final reaction time. (B) Identification of the nucleoside methylated by NS5MTase_DV. RNAs incubated in the presence of Ado[methyl-³H]Met and purified recombinant NS5MTase_DV were treated with phosphodiesterase and alkaline phosphatase, and analysed using thin-layer chromatography. The experiment was performed independently twice. The figure shows a qualitative analysis of one chromatogram. Indicated positions of marker nucleosides [N7-methylated guanosine, (^7MeG), guanosine (G), adenosine (A) and 2′-O-methylated adenosine (A_2′OMe)] were determined under UV light. The radiolabelled products were analysed as described in Materials and methods.

Fig. 6. The binding of GTP by NS5MTase_DV. (A) Cross-linking of [α-³²P]GTP to NS5MTase_DV. NS5MTase_DV was incubated with 50 µM (1 µCi) of either [α-³²P]GTP (lanes 1 and 2) or [α-³²P]ATP (lane 3) in the presence (lanes 1 and 3) or absence (lane 2) of 5 mM Mg²⁺. Bacteriophage T4 DNA ligase (Lig) served as a positive control (lane 4). The upper panel shows the Coomassie Blue-stained gel, and the lower panel the corresponding autoradiographic analysis. (B) Determination of the dissociation constant K_d of [α-³²P]GTP for NS5MTase_DV. Increasing concentrations of [α-³²P]GTP were incubated with NS5MTase_DV, then UV irradiated to form a covalent complex and finally analysed using SDS–PAGE (upper panel) and autoradiography (lower panel). Background radioactivity in lane 0 results from cross-contamination from the adjacent well (not shown). Data points represent bound radioactivity and are the averages of three independent experiments. The K_d was determined using hyperbolic curve fitting. (C) Competition assay for the binding of cap analogues ^7MeGTP, ^7MeGpppA and GpppA, and ATP to wild-type NS5MTase_DV. NS5MTase_DV was incubated with 50 µM radiolabelled GTP and increasing concentrations of competitors. Data points represent the percentage of [α-³²P]GTP-binding to NS5MTase_DV in the presence of a given competitor and are the averages of three independent experiments. Dissociation constants were determined using a descending hyperbolic curve fit.

Fig. 5. Comparative analysis of NS5MTase_DV. (A) Structure-based sequence alignment of the MTase core domain of NS5MTase_DV with rRNA MTase FtsJ (Bugl et al., 2000), the C-terminal rRNA MTase domain of Mj0697 (Wang et al., 2000), mRNA MTase VP39 (Hodel et al., 1996), mRNA MTase domains I and II of Reovirus protein λ2 (Reinisch et al., 2000), the rRNA MTase ErmC′ (Bussiere et al., 1998) and the DNA MTase M.TaqI (Schluckebier et al., 1997). The positions of conserved DNA MTase motifs I to X [except for motif IX, which is only conserved for (cytosine-5-) DNA MTases; Posfai et al., 1989] are indicated in yellow (Malone et al., 1995). In order to simplify the alignment, additional structural elements in the sequences of λ2 MTase II (between residues Ile936 and Ser961 as well as Arg989 and Lys1014) and in M.TaqI (between residues Gly112 and Ala132) were omitted. The omissions are indicated by red circles below the sequences. The alignment was done manually using Seaview (Galtier et al., 1996) according to the superposition of the structures using TURBO (Roussel and Cambillau, 1991), and the final form of the alignment was generated with ESPript (Gouet et al., 1999). The secondary structure elements of the NS5MTase_DV core are given above the alignment and are named as in Figure 1B. The residues within DNA MTase motifs I and III, presenting similarity as detected by ESPript, are shown in red with blue outlines. Positions conserved between NS5MTase_DV, FtsJ, VP39 and λ2 MTase I, which coincide with motifs X, IV, VI and VIII, are marked by a red background where the conserved active-site residues K-D-K-E of 2′OMTases are in white. (B) Stereo view of the superposition of active site residues K-D-K-E of VP39 and NS5MTase_DV. Both structures are shown as sticks. The VP39 structure (PDB accession code 1AV6) is depicted with a RNA substrate (^7MeG-capped RNA hexamer) and AdoHcy. VP39 residues K41-D138-K175-E207, AdoHcy and the RNA substrate are coloured according to atom type (oxygen in red, nitrogen in blue, carbon in yellow, sulfur in green and phosphorous in orange; hydrogens are not shown). The target 2′-OH group of the second nucleotide of the substrate is annotated (only a pppG fragment of the substrate ^7MeGpppGAAAAA is depicted). NS5MTase_DV residues K61-D146-K181-E217 are shown in dark blue, AdoHcy was omitted.

Fig. 1. General features of NS5MTase_DV. (A) Predicted domain structure of Dengue protein NS5. The putative N-terminal methyltransferase domain is shown in grey. The position of the AdoMet-binding motif I (residues 77–86) described by Koonin (1993) is highlighted in black. The region of the C-terminal polymerase domain containing motifs I to VIII of positive-strand RNA virus RNA polymerases (Koonin, 1991) is marked in grey. Motifs A to D, shared by RNA-dependent polymerases (Poch et al., 1989), are shown in black. AdoMet, S-adenosyl-l-methionine; NLS, nuclear localization sequence; Pol, polymerase. (B) Crystal structure of NS5MTase_DV in complex with AdoHcy. A ball-and-stick representation is used for AdoHcy, whereas NS5MTase_DV is drawn as a ribbon. The N-terminal subdomain of NS5MTase_DV (residues 7–54) is coloured red. The core subdomain (residues 55–222) has a typical AdoMet-dependent MTase topology and is coloured yellow. The C-terminal part of NS5MTase_DV (residues 223–267) is coloured cyan. The figure was generated using MOLSCRIPT (Kraulis, 1991) and rendered using RASTER3D (Merrit and Murphy, 1994). (C) Sequence alignment of flavivirus NS5MTases coloured according to the ribbon representation of NS5MTase_DV in (B). NS5MTase domains from Dengue virus type 2 New Guinea isolate (D2V), West Nile virus New York isolate (WNV) and Yellow Fever 17D (YFV) were aligned using Clustal_W (Thompson et al., 1994) and rendered using ESPript (Gouet et al., 1999). Secondary structures (α-helices and β-strands) of subdomains 1, 2 and 3 are indicated above the alignment and coloured in red, yellow and cyan, respectively. Helices and strands are named using Greek letters inside the core domain (subdomain 2), and roman letters outside (subdomains 1 and 3). Amino acids involved in GTP binding (see text) are indicated by a grey star below aligned sequences, and amino acids interacting with AdoHcy are indicated by a pink sphere.