RrmJ has been co-crystallized in the presence of the cofactor AdoMet and its structure has been solved to a resolution of 1.5 Å (Bügl et al., 2000). The crystal structure of RrmJ does not contain the 29 N-terminal amino acids because this part had to be cleaved off in order for RrmJ to crystallize. The crystal structure of RrmJ revealed the classical methyltransferase fold. This common core structure consists of a seven stranded sheet, with the seventh β-strand being antiparallel to the other six and inserted into the sheet between β-strand 5 and 6.
Six α-helices are associated with the β-strands, giving a α / β / α sandwich structure (Fauman, 1999). A special feature of RrmJ’s structure is the extended α4-helix which is seven turns longer than in the consensus structure, which is represented by the catechol-O-methyltransferase COMT (Bügl et al., 2000; Fauman, 1999).
Figure 2: RrmJ has a methyltransferase fold (Bügl et al., 2000)
Stereo diagram of the RrmJ tertiary fold highlighting secondary structure element. Secondary structures were assigned and the figure was rendered by RIBBONS (Carson, 1997). The bound AdoMet is shown in ball and stick representation.
1.3.1 Structurally related methyltransferases
Structure analysis and comparison of RrmJ with other methyltransferase structures (Dali 2.0; Holm and Sander, 1997) revealed five highly similar proteins: catechol-O-methyltransferase (COMT) (Vidgren et al., 1994), Taq I polymerase (Schluckebier et al., 1997), Vaccinia virus mRNA 2’-O-methyltransferase VP39 (Hodel et al., 1996), rRNA methyltransferase ErmC (Bussiere et al., 1998) and 1FBN, the fibrillarin homologue from Methanococcus jannaschii (Wang et al., 2000). Both, COMT and VP39 are single domain structures like RrmJ. VP39 contains a homologous core domain and a substrate binding groove, which is highly similar to the surface of RrmJ (Bügl et al., 2000).
1.3.1.1 VP39
VP39 is a bifunctional protein in Vacchinia virus, which participates in the maturation of both mRNA ends. At the 5’ end, VP39 acts as an AdoMet-dependent cap specific mRNA 2’-O-methyltransferase, catalyzing the ribose methylation of the first transcribed nucleotide.
At the 3’end of the mRNA, the protein acts as a smaller subunit of the heterodimeric Vacchinia virus poly (A) polymerase (Hodel et al., 1996).
VP39 has been co-crystallized in the presence of an mRNA substrate analog, a m7(5’)Gppp-capped single stranded RNA hexamer and the competitor Adenosine-L-homocysteine (AdoHCys). It comprises a compact single core domain with the typical α/β/α fold (Hodel et al., 1996). Surface comparison between VP39 and RrmJ revealed striking similarities (Fig. 2) (Staker, 2000). A significant feature in both RrmJ and VP39 is the extended α4-helix located next to the AdoMet binding site and above the substrate binding site of VP39. The VP39 mRNA binding groove strongly resembles a highly conserved surface area of RrmJ in both size and distribution of positively charged residues (Staker, 2000). A structure based sequence alignment showed 18 identical amino acids in VP39 and RrmJ (Staker, 2000). Modeling studies revealed that VP39’s mRNA substrate fits nicely into the putative substrate binding site of RrmJ (Staker, 2000). Four of the six amino acid residues in VP39, which have direct contact to the mRNA substrate, are structural identical in RrmJ.
All four are highly conserved amino acids and are Lys41 (Lys38), Lys175 (Lys164), Ser205 (Ser197) and Glu207 (Glu199) (VP39 residues are in italics). In VP39, they form a hydrogen bond network that coordinates the phosphate atoms on each side of the methylated nucleoside (Bügl et al., 2000).
Enzymatic characterizations of purified VP39 revealed that this enzyme has a Kcat of 0.13 min-1 and therefore, a similarly low turnover rate than RrmJ (Hu et al., 2002). In addition, the Km value for the cofactor AdoMet (2 µM) was similar to the Km value that was observed for RrmJ (3.7 +/- 0.3 µM) (Barbosa and Moss, 1978; Hager et al., 2002). VP39’s Km value determined for the mRNA substrate (5 nM) was found to be significantly lower compared to RrmJ’s Km value for 23S rRNA (0.8 +/- 0.1 µM) (Barbosa and Moss, 1978;
Hager et al., 2002). This, however, might simply reflect the abundance of the respective substrates in the cell, where only 5-10% of total RNA is mRNA, but 75-80% of total RNA is rRNA (Knippers, 1995).
Figure 3: Comparison of RrmJ and VP39 Surfaces (Bügl et al., 2000)
(A) Solvent contact surface of RrmJ. The electrostatic potential is shown by the color code: -15 (blue), 0 (white) and +15 KbT (red). The bound AdoMet is shown in stick presentation. (B) Solvent contact surface of VP39 and its electrostatic potential. The bound AdoHCys is shown. It is identical in orientation, scale and coloring to RrmJ in (A). (C) The 27 most conserved RrmJ residues are colored green. (D) The putative substrate binding groove of RrmJ is shown. The amino acids that might be important for substrate binding are indicated.
1.3.1.2 Fibrillarin
A second structurally similar enzyme of RrmJ is the Methanococcus jannaschii fibrillarin 1FBN, whose structure has been solved to a resolution of 1.6 Å (Wang et al., 2000).
Comparison of the two proteins revealed that RrmJ and fibrillarin share 19% sequence identity and 154 amino acids between RrmJ and fibrillarin superposed with an rmsd of 2.6 Å3 (Staker, 2000). In contrast to RrmJ, however, fibrillarin consists of a globular two-domain structure, built of a smaller N-terminal two-domain and a larger C-terminal two-domain.
While the N-terminal domain represents a novel fold, the core of the connected C–terminal domain shows the classical methyltransferase fold (Wang et al., 2000). The crystal structure
revealed that the two N-terminal domains of fibrillarin join together to form a homodimer.
Because fibrillarin was crystallized without bound AdoMet, the exact position of the cofactor is not known. Also methyltransferase activity has not yet been detected in vitro and a complete biochemical characterization of fibrillarin remains to be performed (Wang et al., 2000). However, based on the structural analysis and the association of fibrillarin with box C/D snoRNAs, it has been suggested that fibrillarin functions as rRNA 2’O-methyltransferase. The homology to RrmJ furthermore supports this hypothesis.
In yeast, several temperature sensitive mutants of the fibrillarin homologue Nop1p have been isolated (Tollervey et al., 1991; Tollervey et al., 1993). Nop1p functions in all three major posttranscriptional activities; i) it takes part in the cleavage of the large pre- ribosomal RNA into the mature 18S, 5.8S and 25S RNAs, ii) it is involved in 25S modification by methylating and pseudouridylation of the rRNA sequences and iii) it takes part in the assembly of the ribosomes (Tollervey et al., 1993). Some of these Nop1p mutants show severe ribosome assembly defects as well as significantly impaired growth. In order to understand the effects of the Nop1 mutants better, the mutations were localized in the structure of fibrillarin and compared to the structure of RrmJ (Staker, 2000). In this way residues in Nop1 could be identified which might be involved in AdoMet binding and therefore, the effect of four of the Nop1 mutations could be attributed to impaired AdoMet binding (Staker, 2000).