In the course of a large-scale analysis of MTase sequences and structures we observed low similarity between various ribose 2'-O
-MTases from eubacteria and negative-strand RNA viruses and a fragment of the grass carp reovirus protein [13
] corresponding to the carboxy-terminal part of MTase 1 of human reovirus (J.M.B. and L.R., unpublished data). This prompted us to reanalyze the sequences and structures of the reovirus MTase domains. Disappointingly, sequence database searches carried out using PSI-BLAST [14
] initiated with the λ2 sequence and its fragments revealed significant similarities only between the human reovirus and grass carp reovirus proteins. Nonetheless, VAST [15
] searches of the Protein Data Bank [16
] revealed high similarity of both putative MTase domains to numerous known MTase structures. Detailed inspection of the coordinates superimposed using Swiss-Pdb Viewer [17
] allowed us to predict which amino-acid residues might be involved in cap 0 and cap 1 methylation of reovirus mRNA.
According to VAST, MTase 1 showed highest similarity to the RrmJ (1ejo; P
-value = 10-10.4
) and VP39 (1av6; P
-value = 10-8.1
) proteins. The mutual similarity of MTase 1 and MTase 2, which can be regarded as a reference, was evaluated as high, albeit significantly lower (P
). RrmJ and VP39 are both 2'-O
-ribose MTases; RrmJ targets U2552 in eubacterial rRNA [18
], whereas VP39 is a bonafide
cap 1 MTase of vaccinia virus mRNA [20
]. Remarkably, simultaneous superposition of the three structures revealed perfect conservation of the K-D-K-E tetrad of putative catalytic residues (Figures ,,), which has been observed by us in other families of genuine and putative 2'-O
] and J.M.B. and L.R., unpublished data). These results strongly argue that MTase 1 is more likely to be a cap 1 MTase than a cap 0 MTase.
Figure 1 Stereoview ribbon diagrams of superimposed structures of the MTase 1 domain (green), VP39 (blue) and RrmJ (magenta). MTase 1 shows elaborations of the common fold similar to those of VP39, which are altogether absent from the RrmJ structure and which (more ...)
Figure 2 Sequence alignment of the predicted human reovirus cap 1 MTase with its counterpart from grass carp reovirus (GCRV) and vaccinia virus (VP39). Residues that are invariant between the reoviruses and those predicted to participate in binding of the AdoMet, (more ...)
Figure 3 Stereoview of superimposed structures of the MTase 1 domain (ribbon in green, side chains and labels in red) and VP39 (ribbon and labels in blue, side chains in cyan), delineating the proposed binding sites for the guanine moiety and the target ribose (more ...)
In the MTase 1 structure we could identify no aromatic residues that would superimpose well with Y22 and F180 from VP39, which form enhanced stacking interactions with the N7-methylated cap [20
]. Moreover, a region corresponding to the guanine-binding pocket in VP39 is blocked off by a differently positioned loop in the reovirus λ2 protein structure. Nevertheless, we identified a cluster of aromatic side chains (Y460/464, F461/465 and F618/F622) that are invariant between the human and grass carp reovirus sequences and map in the vicinity of the methylated cap, if the mRNA coordinates are copied from the superimposed VP39 structure (Figure ). It is tempting to speculate that a conformational change in this region occurring on mRNA binding would, for instance, reallocate Y460 and F618 residues of human reovirus to positions equivalent to Y22 and F180 from VP39. The sequence alignment (Figure ) has been manually adjusted to illustrate this hypothesis.
The MTase 2 structure showed highest similarity to the glycine N
-MTase (GNMT; 1xva; P
-value = 10-10.1
). It is noteworthy that the MTase 2 domain showed lower similarity (P
-value = 10-4.3
) to VP39 than to GNMT and the MTase 1 domain showed lower similarity (P
-value = 10-7.9
) to GNMT than to the 2'-O
-ribose MTases. Figure shows that in addition to the common catalytic domain, GNMT and MTase 2 share a topologically equivalent 'lid' domain made of three antiparallel β strands. In GNMT, the β-lid domain forms a wall of a large 'molecular basket' structure, which may accommodate a variety of small molecules, including AdoMet, tetrahydrofolate and polycyclic aromatic hydrocarbon molecules such as benzopyrene (reviewed in [22
]). In MTase 2, the strands forming the β-lid domain are much shorter and the pocket is also smaller. However, a guanine moiety can be docked into that pocket in a way that the N7 nitrogen is presented for the methyl group donor (data not shown). Most of the amino acids that line up the putative guanine-binding pocket of MTase 2 (for instance Q925, N927, F951, R956, and E958) are conserved between the human and grass carp reovirus sequences (Figure ).
Stereoview of superimposed structures of the MTase 2 domain (green) and GNMT (blue) showing extensive similarities of both domains.
Sequence alignment of the MTase 2 domain with its counterpart from grass carp reovirus. Invariant residues are highlighted in black, conservatively substituted residues are highlighted in gray. Motif nomenclature follows .
To our knowledge, all structurally characterized MTases that modify bases in nucleic acids and do not employ covalent bond formation with the target use aromatic or aliphatic side chains to bind the base to be methylated and stabilize it in the active site. Examples of such MTases, in which the structure of the active site was determined experimentally or predicted from sequence analysis, include enzymes generating N6
-methyladenine in DNA and RNA (reviewed in [23
-methylcytosine in DNA [24
], and N2
-guanine in RNA [25
]. We hypothesize that the invariant F951 and L890 (substituted by F897 in grass carp reovirus) may be involved in van der Waals interactions with an aromatic ring of guanine. But because of the low resolution of the original structure and lack of a precise docking model, the detailed contacts between the target and the enzyme could not be predicted. Nevertheless, we believe that our model will be a useful guide for site-directed mutagenesis experiments that would allow to elucidate the role of individual side chains of the putative cap 0 MTase.