Functional characterization of BsTrmB
The ytmQ ORF in B.subtilis encodes an ortholog of the E.coli MTase TrmB responsible for the formation of m7G46 in the variable loop of tRNA. To test the MTase activity of the ytmQ gene product the purified protein was incubated with [methyl-14C]AdoMet and T7 in vitro transcribed B.subtilis tRNAPhe. The tRNA was subsequently hydrolyzed by nuclease P1 and the resulting 5′-phosphate nucleosides were analyzed by bidimensional cellulose thin layer chromatography (2D-TLC) followed by autoradiography. reveals the formation of a radioactive compound with the migration characteristics of 7-methylguanosine 5′-phosphate (pm7G). On the other hand, radioactive (32P) B.subtilis tRNAPhe transcripts were obtained by T7 in vitro transcription in the presence of [α-32P]GTP or [α-32P]UTP. These transcripts were incubated in the presence of the purified ytmQ gene product and non radioactive AdoMet. After incubation, the [α-32P]GTP-labelled transcript was hydrolyzed by nuclease P1 (generating 5′-phosphate nucleosides) and the [α-32P]UTP-labelled transcript was hydrolyzed by RNAse T2 (generating 3′-phosphate nucleosides-nearest neighbor analysis: B.subtilis tRNAPhe contains U47). The hydrolysates were analyzed by 2D-TLC followed by autoradiography. The formation of a modified guanosine with migration characteristics of pm7G was observed in the case of [α-32P]GTP-labeled tRNAPhe (data not shown) whereas the formation of a nucleotide with migration characteristics of 7-methylguanosine 3′-phosphate (m7Gp) was observed with [α-32P]UTP-labeled tRNAPhe (). These results show that the B.subtilis ytmQ gene product displays the same activity as the E.coli TrmB enzyme, generating m7G at position 46 in tRNA. For this reason, the ytmQ gene has been renamed trmB and the gene product BsTrmB.
Figure 1 Affinity-purified ytmQ gene product (BsTrmB) catalyzes the formation of m7G in T7 transcripts of B.subtilis tRNAPhe. (A) B.subtilis tRNAPhe (5 µg) was incubated with 5 µg of purified BsTrmB protein and 15 µM of [methyl-14C]AdoMet (more ...)
The B.subtilis strain BFS1045, in which the trmB gene is inactivated by an insertion, shows normal growth, demonstrating that neither the BsTrmB protein, nor the m7G46 modification are essential. Total (bulk) tRNA extracted from the BFS1045 strain was shown to be a substrate for the purified BsTrmB enzyme ().
Figure 2 Total tRNA extracted from the the B.subtilis strain BS1045 with a disrupted trmB gene is a substrate for the affinity-purified BsTrmB MTase. Total tRNA (5 µg) was incubated with the purified BsTrmB enzyme (5 µg) in the presence of 30 µM (more ...)
The formation of a specific complex between BsTrmB and a tRNA substrate was tested by the electrophoretic mobility shift assays in the presence or absence of sinefungin, a cofactor analog. As can be seen in , a bandshift is observed in the presence of sinefungin, indicative of complex formation. When very high enzyme/substrate ratios are used, higher molecular mass aggregates appear, even in the absence of sinefungin. Experiments are in progress to determine the stoichiometry of the complex between BsTrmB and the tRNA.
Figure 3 Complex formation between BsTrmB and B.subtilis tRNAPhe transcript. The experimental conditions are described in Materials and Methods. The enzyme to substrate ratio (E/S) varies from 3 to 10. The experiment was performed with or without addition of sinefungin (more ...)
Overall structure of BsTrmB
The crystals of BsTrmB diffracted to 2.1 Å, but the structure could not be fully traced automatically with the program Solve/Resolve. Therefore most of the structure was built manually. The electron density for the two BsTrmB molecules in the asymmetric unit is clear for residues 10–210. The 10 N-terminal residues of the protein and the His-tag are not visible in the electron density. Analysis of the Ramachandran plot (Supplementary Figure 1) revealed that 95.8% residues are in favored regions, and 99.0% residues are in allowed regions. There are four outliers (E86 and L185 in monomer A and G187 and S198 in monomer B), all in surface-exposed regions with a relatively high temperature factor. The 3D fold of the BsTrmB monomer is very similar to the RFM structure, also termed ‘class I’ MTase fold (34
) ( and ). It consists of a seven-stranded β-sheet flanked by helices, and differs from the canonical Rossmann-fold by an additional strand inserted between strand 5 and 6 (35
). Most parts of the structure of BsTrmB (including the conserved motifs I-VIII) are in general agreement with the theoretical model we proposed previously for the orthologous EcTrmB enzyme (24
). Nonetheless, the crystal structure of BsTrmB reveals one striking difference with respect to all previously characterized members of the RFM fold that was also unaccounted for in the previous model of EcTrmB: residues 10–40 of the N-terminus fold back over the protein, over helix C, strand 2 and 3, and helix B. As a result, the N-terminal helix (corresponding to motif X) is located on an opposite part of the structure than in other RFM enzymes ().
Figure 4 (A) Overall view of the dimer structure of BsTrmB. (B) More detailed view of the dimer interface, with the potassium ion shown in magenta and water molecules in red. (C) Ribbon drawing of BsTrmB monomer A with the positively charged residues in loop 121–128 (more ...)
Figure 5 Topology diagram of typical Rossmann-fold dehydrogenases, Class-I (RFM) MTases and TrmB. Triangles indicate β-strands in parallel orientation, the inversed triangles in MTases indicate the only antiparallel strand, circles indicate helices, connectors (more ...)
Searches in the PDB for structures related to BsTrmB revealed that a closely related structure from Streptococcus pneumoniae (here termed SpTrmB) was solved by a structural genomics consortium, but not yet analyzed in the literature (1YZH; Y. Kim, H. Li, F. Collart and A. Joachimiak, manuscript in preparation). SpTrmB and BsTrmB exibit the 0.89 Å average r.m.s.d. between 190 superimposable Cα atom pairs and share the unusual conformation of the N-terminal region. The second best matches for BsTrmB in the PDB are the more remotely related AdoMet-dependent methyltransferase from Mycobacterium tuberculosis (1I9G, RMSD 2.2 Å for 141 Cα atom pairs) and catechol O-MTase (1VID, RMSD 2.2 Å for 141 Cα atom pairs), one of the smallest class I MTases, which exhibits an orthodox RFM fold.
The BsTrmB structure contains three potassium ions per asymmetric unit, one in the interface between monomers and one in a region involved in AdoMet binding in the homologous catechol O-MTase, bound to the main chain carbonyls of residues Asn115 and Gly46.
In the crystal BsTrmB forms a homodimeric structure, with two molecules per asymmetric unit. The dimer surface covers helix E (residues 154–170), and strand 6 (172–179), and buries a surface of 805 Å2 per molecule. The dimer contact buries a cluster of five water molecules of which three are bound to a potassium ion. The potassium ion itself is bound to residues Ser167 OH from molecules A and B, and to the main chain carbonyls of Leu171 from molecules A and B. It is also coordinated to Arg212 NH2 from molecules A and B, at a distance of 4.0 Å. The rest of the interface consists largely of hydrophobic interactions involving Phe159, Leu163, Leu173, Leu176 and Leu178, and hydrogen bond contacts involving residues Ser183, Arg212 and Arg156 on the edges of the interface (). The analysis of the molecular mass of the BsTrmB by gel filtration studies confirms that BsTrmB is dimeric also in solution, and that dimerization is not a consequence of crystal packing only ().
Figure 6 Gel filtration analysis of BsTrmB. Molecular mass determination was performed using an Amersham Biosciences Superose P12 HR (1 × 30 cm) column equilibrated with 50 mM Tris–HCl, pH8, 10 mM MgCl2, 300 mM KCl and 10% Glycerol. The flow rate (more ...)
Analysis of the asymmetric unit of the SpTrmB structure (1YHZ) reveals two molecules, however in an orientation that is unlikely to be biologically relevant (low interface ASA and lack of features typical for real homodimers—data not shown). Thus, symmetry related molecules were created by applying symmetry operators from space group C121 in which SpTrmB crystallized. In the reconstructed unit cell we identified a putative biological interface essentially identical to that in the BsTrmB dimer. All other possible crystal interfaces in SpTrmB have very small interface ASA or big gap volume (data not shown) and were regarded as resulting from crystal packing.
The common interface of BsTrmB and SpTrmB dimers was analyzed in respect to the physical and geometrical properties and compared with values obtained for homodimers (27
) ( and ). It was shown previously (21
) that the combination of these parameters (Materials and Methods) can be successfully used to discriminate between crystallographic interfaces and biological interfaces with very low false-positive rate (0.6%). Most homodimers have Fbu
> 30%, Fnp*
B > 800 Å2
and RP > 0. Dimeric interfaces of BsTrmB and SpTrmB have very similar features to those typical for homodimers (). However, a mapping of evolutionary rates () onto the surface of the structure and statistical test of a significance of a conservation level of the interface show that the interface is not as conserved as expected for homodimers. The level of conservation numerically is described with P
-value equal to 0.35 which does not allow to reject a null hypothesis that the interface is not more conserved that the rest of the surface. Small interface area, low RP score and moderate level of conservation suggest that either BsTrmB and SpTrmB belong to the class of transient homodimers (36
) or the dimer structure is only conserved within the subgroup of TrmB enzymes from Bacilli
Analysis of interfaces in 1yzh and ytmq in terms of conservation of interfaces
Analysis of the dimer interfaces in Bs and SpTrmB
Figure 7 Evolutionary rates derived from Bs and SpTrmB (A) and other TrmB-like sequences (B) mapped on the surface of BsTrmB structure. Gradient of colors represents the change of conservation: from red (not conserved) to blue (conserved). The black ribbon represents (more ...)
The dimerization interface is formed by a central hydrophobic cluster of five residue pairs surrounded by a polar and charged rim of partially buried residues (). The hydrophobicity of the central region is conserved in BsTrmB and SpTrmB but not in all TrmB MTases. In particular, the region consists of Phe159 and Leu173 (hydrophobicity conserved only in Bs-like TrmBs), Leu163 (hydrophobicity conserved both in Bs- and Ec-like TrmBs), Leu176 [hydrophobicity conserved only in Bs-like TrmBs (except Mollicutes)], Leu 178 [hydrophobicity conserved in BsTrmB-like MTases but only in one subgroup (mainly Bacillales)].
Surface representations of BsTrmB (A) and SpTrmB (B) structures colored by atomic type (red, oxygen; blue, nitrogen; white, carbon). The black ribbon represents beta strand 6 and alpha helix E of the second subunit of a dimer.
The rim of polar residues in the interface is only moderately conserved. It seems that this region can accept substitutions of several polar amino acids without losing the ability to dimerize. The interaction in the rim region is formed by a complicated network of hydrogen bonds and water bridges. Not all hydrogen bonds and water bridges are preserved in crystal structures of BsTrmB and SpTrmB despite their sequence similarity (). Apparently, this network of hydrogen bonds and electrostatic interactions is even less similar in more distantly related BsTrmB-like MTases where it is created by different interacting amino acids.
Figure 9 Surface representations of BsTrmB monomer (A) colored by atomic type (red, oxygen; blue, nitrogen; white, carbon) and BsTrmB dimer (B) colored by chain (chain A, green; chain B, orange). Red and yellow overlapping balls represent conserved positions of (more ...)
Identification of the ligand-binding and active sites
At present only the structure of the ligand-free BsTrmB is available. However, from the homology to other RFMs, the knowledge that AdoMet acts as a co-factor in the tRNA (m7
G46) methylation reaction, and the mutagenesis data obtained for the orthologous EcTrmB enzyme (24
) it is possible to identify the functionally important sites.
RFMs bind the methyl donor AdoMet in a deep groove formed by the C-terminal edges of strands 1–3. This is the most conserved region in the RFM superfamily, both at the sequence and structure level. The comparison of BsTrmB to the structure of catechol O-MTase (COMT) complexed with AdoMet () shows conservation of the AdoMet-binding residues typical for motifs I, II and III. We carried out computational docking of AdoMet to the BsTrmB structure, which revealed that the preferred binding of the cofactor is similar to that observed in other members of the superfamily (Supplementary Data). According to the sequence conservation and the docking model, Glu44 is inferred to coordinate the methionine moiety, Glu69 may coordinate the ribose hydroxyl groups, and Asp96 may coordinate the N6 group of the adenine moiety.
On the other hand, the sequences and structures of the substrate-binding/active site are not conserved or only weakly conserved between different families of MTases (and this is also the case between BsTrmB and COMT). In BsTrmB, a large insertion (residues 179–200) folds over the active site region. This insertion is located between strands 6 and 7, in a site where structurally variable insertions are frequently found among different RFMs. The electron density is relatively weakly defined in the hinge regions around the insertion in molecule A and in the whole insertion in molecule B. The insertion is anchored to the rest of the protein mostly by hydrogen bonds, and a few hydrophobic residues (Tyr193 and Phe197) that cluster with Phe116 and Ile204. Tyr 193 is conserved (or conservatively substituted by Phe) in the TrmB family and is positioned in such a way that it could stack with a guanine bound in the active site in BsTrmB, e.g. in analogy to the role fulfilled by Val21 in the m6
A MTase M.TaqI, the only MTase acting on purines in nucleic acids that has been successfully crystallized with the substrate in the active site (37
The invariant Asp154 and conserved Thr153 could be involved (directly or indirectly) in coordination of the N2
amino group and the O6
group, respectively, of the target guanine base. In agreement with this finding, the mutation of Asp180 in EcTrmB (homologous to Asp154 in BsTrmB) abolishes tRNA binding, but has only a minor effect on AdoMet binding (24
Using SURFLEX and GRAMM we constructed a preliminary computational docking model of the BsTrmB-AdoMet-tRNAPhe
complex. The model (; coordinates available as Supplementary Data) suggests an overall good surface complementarity between the protein and the tRNA molecules. However, the methylated N7
atom of the target guanosine is turned away from the methyl group of AdoMet and if our model is correct, then a significant conformational change of the tRNA substrate would be required to flip G46 into the active site of the enzyme. Interestingly, the same binding mode is equally compatible with the ‘lambda’ form of the tRNA previously observed in the archaeosine tRNA-guanine transglycosylase (7
), where the D-arm unfolds and loses its tertiary interactions with the tRNA core. A similar rearrangement in the context of our model would result in clearing the space for G46 and enabling its rotation into the catalytic site (data not shown). Unfortunately, the computational simulation of such rearrangement is beyond the limits of the available methods. The current docked model remains to be tested experimentally (for instance by cross-linking experiments), both with respect to the mutual orientation of the protein and the tRNA, and the type of the potential conformational change and base-flipping of G46.
Figure 10 A computational docking model of AdoMet (yellow spheres) and tRNA (white sticks) to the BsTrmB dimer (in green, one monomer shown using the surface representation, the other as cartoons depicting secondary structures). The target guanosine in the original (more ...)
Importantly, the crystal structure of BsTrmB allows us to revise the role of conserved Thr191 and Glu194 residues, whose homologs in EcTrmB (Thr217 and Glu220) were found to be important for the enzyme activity and predicted to be involved in tRNA binding. Thr191 is located at the bottom of the AdoMet-binding pocket, where it is predicted to participate in the stabilization of the methionine moiety, while Glu220 is positioned away from any possible binding sites and seems to be involved in the stabilization of the 179–200 insertion.
The 179–200 insertion in BsTrmB occupies the space that in other RFMs (e.g. in the aforementioned COMT or M.TaqI) is filled by the N-terminal helix. Thus, it appears that TrmB evolved a novel structural element involved in the binding of the target base, which displaced (both in the functional and structural sense) the N-terminal region typically used for this purpose. The N-terminal region was relocated to the completely opposite part of the structure. This finding is important for the evolutionary studies of the RFM superfamily and in general, provides a new example of how the protein folds can change in the course of the evolution (38
Part of the putative substrate-binding site of BsTrmB is a loop containing the highly positively charged sequence 121-PKKRHEKR-128. The electron density of this loop is very clear despite the fact that it protrudes quite far out of the protein surface. The location near the active site region, and the large accessibility and concentration of positively charged residues makes this loop a candidate for involvement in the binding of the tRNA substrate. In our docking model this loop makes extensive contacts with the tRNA. Indeed, for the homologous EcTrmB enzyme it was shown that mutations of residues homologous to His125, Arg128 or Arg129 (His151, Arg154A and Arg155A, respectively) to alanine all reduced the activity of the enzyme to below 10% of the wild-type activity, and the mutant Arg150A (residue homologous to Arg124 in BsTrmB) had only 30% of the wild-type activity. The mutation Asn152A in EcTrmB (the equivalent of Glu126 in BsTrmB) did not have any impact on catalysis or tRNA binding. In agreement with this finding, the side chain of Glu126 in the structure of BsTrmB points away from the potential tRNA-binding site.