Table 1.
Crystal, collection, structure determination data and refinement statistics.
Figure 1.
Cartoon representation of the nsp10/nsp16 complex with the reaction product SAH and metal ions.
A) The nsp16 protein (green) is bound to nsp10 (yellow) through an interface which does not involve Zn ions (grey spheres) present in nsp10. One metal ion (blue sphere) is found in nsp16 on the opposite face from the active site to which a SAH molecule is found (red sticks). B) Ribbon representation of nsp16, rainbow colors from N- to C-terminus. Top: Each secondary structure element is labeled according to[25] (see also Fig. S3). The SAH molecule shown in sticks colored following atom type. Bottom: View of interface involving nsp16 (green surface) and nsp10 (yellow ribbons) showing the nsp10 secondary structure elements involved in the interface.
Figure 2.
Topology diagram of MTase secondary structure elements.
A) The consensus topology diagram is shown with a two domain organization for methyl acceptor substrate and SAM, as defined[25] with the catechol O-MTase and its canonical seven strand beta sheet. B) Topology diagram of nsp16 colored according to rainbow colors from N-to C-terminus as in Fig. 1B. The missing helix B and helix C are indicated by dashed ovals. The approximate general binding site of nsp10 is shown (yellow oval).
Figure 3.
The SAM binding site of nsp16.
A) SAH modeled in a simulated annealing Omit map contoured at 1σ. Carbons, oxygens, sulfur, and nitrogens are in grey, red, yellow, and blue sticks, respectively. Water molecules are shown as red spheres. B) Sinefungin bound in the SAM/SAH binding site, with main catalytic residues and the water molecule indicative of a catalytic mechanism. Colors as in A). C) SAH modeled in a 2Fo-Fc map (green) of Sinefungin contoured at 1σ with a Fo-Fc difference map (red) contoured at 3σ. A peak of negative density appears clearly on the sulfur atom of the SAH molecule showing that SAH was indeed replaced by Sinefungin. Colors as in A).
Table 2.
Mutational analysis, complex formation, and enzyme activity of the nsp10/nsp16 complex.
Figure 4.
Position and coordination of a metal ion.
Left, the putative Mg2+ ion (blue sphere) is shown solvated in its first atomic shell by six water molecules (red spheres). The corresponding 2Fo-Fc electron density map (contoured 1σ) with a cross shape is shown in purple. Residues of Nsp16 involved in coordination of Mg2+ ion via water molecules are labeled. Right, a global view of the bound metal ion in its electron density on the opposite side from the SAH molecule (red sticks). Nsp16 is shown as green ribbons, nsp10 as yellow ribbons with its two bound Zn2+ as grey spheres as in Fig. 1.
Figure 5.
Stick model of RNA bound to the nsp16 RNA binding groove and Sinefungin in the methyltransferase active site.
A) In this representation, Sinefungin was preferred over SAH because one of its NH2 groups approximates the direction of a transferred CH3 group from the SAM substrate. Carbon is white, oxygen is red, nitrogen is blue and phosphorous is orange. Nsp16 and nsp10 are rendered as a solvent-accessible surface colored grey and wheat respectively. The Sinefungin molecule defines the methyltransferase active site. Missing residues in the 135-137 loop (see text) are indicated by a shaded blue dotted box. Position of Y30 and Y132 are indicated. Y30 generated poor electron density (see « Methods ») and its aromatic ring position has been manually adjusted before generation of this image. B) Close caption of the methyltransferase active site showing distance between the NH2 of Sinefungin to the 2′-O of the ribose of the first base, thus mimicking the position of the methyl of the S-adenosylmethionine.
Figure 6.
Detailed definition of nsp16 and nsp10 elements involved in the interface.
A) nsp16 (left) and nsp10 (right) are rendered as a solvent-accessible surface colored green and yellow, respectively. Residues involved in the interface are rendered as sticks with the same color code, in transparency into respective proteins. B) Separate representation of the nsp16 (left) and nsp10 (right) interface. Nsp16 residues defining patch I, II, III, and IV are rendered as sticks as above in transparency into nsp16. Nsp10 residues defining patch A, B, C, D, and E are rendered as sticks as above in transparency into nsp10. C) A plot of MTase activity as a function of nsp10/nsp16 complex formation using the data reported in Table 2.