Structure-based identification of functional residues in the nucleoside-2'-O-methylase domain of Bluetongue virus VP4 capping enzyme

Stewart, Meredith E.; Roy, Polly

The 2.5 Ã… crystal structure of the 74 kDa BTV VP4 suggested that residues 175 to 377 encode a distinct 2 0 -O MT domain segregated from the other domains and is responsible for methylation of cap0 structure (Fig. 1) . Currently, there is no direct evidence available that this domain is indeed responsible for 2 0 -O MTase activity, although it includes putative signature catalytic residues, K178-D265-K306-E335, similar to the other catalytic motif of class I AdoMet-dependent methyltransferases ( Fig. 1; [33] ). Based on previous VP4 ligand binding data [33] and studies on other viruses, we hypothesized that aspartic acid 265 (D265) is critical for 2 0 -O MTase activity. We focused on D265 rather than other catalytic residues as it is in proximate distance with the residues that bind S-adenosyl-L-homocysteine (SAH) and guanosine of the cap structure (Fig. 1 ). In addition, we identified a cluster of surface exposed amino acids N311, Y334 and R367 that are in close proximity to the guanosine in the ligand binding pocket ( Fig. 1 ; [33] ). In particular, Y334 and possibly N311 are predicted to interact with guanine of the cap0 [33] , while R367 is believed to be responsible for recruiting the N7 cap ( Fig. 1 ; [33] ). Thus, these residues could be important in assisting the 2 0 -O MTase catalytic activity either through direct interaction with cap0 or by recruiting it within the catalytic domain. Site-specific mutations into the coding region of VP4 were introduced to generate D265E and D265V to either conserve the charge or change the polarity of the residue. Similarly, residues N311, Y334 and R367 were mutated to an alanine either singly or in combination (NYR). The mutated constructs were expressed using the baculovirus expression system and each protein was purified in soluble fraction indicating that the specific mutations, in particular, the surface exposed substitutions, have little or no effect on solubility (data not shown).