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RNA:(guanine-N2) methyltransferases RsmC/RsmD and their homologs revisited--bioinformatic analysis and prediction of the active site based on the uncharacterized Mj0882 protein structure.

Bujnicki JM, Rychlewski L - BMC Bioinformatics (2002)

Bottom Line: Based on the results of sequence analysis and structure prediction, the residues involved in cofactor binding, target recognition and catalysis were identified, and the mechanism of the guanine-N2 methyltransfer reaction was proposed.Using the known Mj0882 structure, a comprehensive analysis of sequence-structure-function relationships in the family of genuine and putative m2G MTases was performed.The results provide novel insight into the mechanism of m2G methylation and will serve as a platform for experimental analysis of numerous uncharacterized N-MTases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bioinformatics Laboratory, International Institute of Cell and Molecular Biology, ul, ks, Trojdena 4, 02-109 Warsaw, Poland. iamb@wp.pl

ABSTRACT

Background: Escherichia coli guanine-N2 (m2G) methyltransferases (MTases) RsmC and RsmD modify nucleosides G1207 and G966 of 16S rRNA. They possess a common MTase domain in the C-terminus and a variable region in the N-terminus. Their C-terminal domain is related to the YbiN family of hypothetical MTases, but nothing is known about the structure or function of the N-terminal domain.

Results: Using a combination of sequence database searches and fold recognition methods it has been demonstrated that the N-termini of RsmC and RsmD are related to each other and that they represent a "degenerated" version of the C-terminal MTase domain. Novel members of the YbiN family from Archaea and Eukaryota were also indentified. It is inferred that YbiN and both domains of RsmC and RsmD are closely related to a family of putative MTases from Gram-positive bacteria and Archaea, typified by the Mj0882 protein from M. jannaschii (1dus in PDB). Based on the results of sequence analysis and structure prediction, the residues involved in cofactor binding, target recognition and catalysis were identified, and the mechanism of the guanine-N2 methyltransfer reaction was proposed.

Conclusions: Using the known Mj0882 structure, a comprehensive analysis of sequence-structure-function relationships in the family of genuine and putative m2G MTases was performed. The results provide novel insight into the mechanism of m2G methylation and will serve as a platform for experimental analysis of numerous uncharacterized N-MTases.

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Comparison of positions of the ligands in the active site of M.TaqI and the docked model of Mj0882. M.TaqI is shown in yellow, Mj0882 is shown in magenta.
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Figure 4: Comparison of positions of the ligands in the active site of M.TaqI and the docked model of Mj0882. M.TaqI is shown in yellow, Mj0882 is shown in magenta.

Mentions: Docking of the target guanine to the Mj0882 structure (Figure 4) was guided by superposition of the "motif IV loop" of Mj0882 and M.TaqI, the only N-MTase co-crystallized with the nucleic acid substrate [29]. Under the assumption that both enzymes bind their targets in the same plane, the N2 group of guanine could be aligned with the N6 group of the adenine amino only if the purine ring was rotated by 120 degrees (i.e. with the atoms C2 and N3 of guanine superimposed onto atoms C6 and N1 of adenine). According to AutoDock [27] the energy of the interactions between the Mj0882-AdoMet complex and the docked guanine is quite favorable and comparable with that calculated for the target adenine in the M.TaqI structure (1g38) (-6.14 kcal/mol and -7.59 kcal/mol, respectively). These values are much lower than the energy of interactions between the protein and the cofactor (see above), because the AdoMet-binding groove is very deep and hydrophobic, while the base-binding site is relatively shallow. An alternative orientation of the target guanine in complex with 1dus could be obtained if its atoms C2 and N1 were superimposed onto atoms C6 and N1 of adenine. However, the latter model resulted in steric clashes between the ribose moiety and I132 (data not shown). Even if the flexible ligand docking option of AutoDock was used, which alleviated steric clashes, energies for the guanine in the "alternative" position (a cluster of similar conformations) were significantly worse (data not shown). In the first docked model the guanine moiety fits the groove on the protein's surface surprisingly well; a residue was identified, whose side chain might coordinate the ribose hydroxyl groups, namely D41 (Figure 3). Analysis of the multiple sequence alignment (Figure 1) revealed that this residue is highly conserved in the Mj0882, RsmD, RsmC, and YbiN families, supporting its functional importance. In the "alternative" docked model, D41 is too remote from the target base to make direct contacts to the target nucleoside (data not shown). However, a substantial conformational change upon substrate binding cannot be excluded, and direct participation of D41 in catalysis as well as other modes of guanine binding can be envisaged. Identification of this conserved carboxylate as a potentially important residue will hopefully prompt site-directed mutagenesis experiments and experimental determination of its role in binding and/or catalysis.


RNA:(guanine-N2) methyltransferases RsmC/RsmD and their homologs revisited--bioinformatic analysis and prediction of the active site based on the uncharacterized Mj0882 protein structure.

Bujnicki JM, Rychlewski L - BMC Bioinformatics (2002)

Comparison of positions of the ligands in the active site of M.TaqI and the docked model of Mj0882. M.TaqI is shown in yellow, Mj0882 is shown in magenta.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC102759&req=5

Figure 4: Comparison of positions of the ligands in the active site of M.TaqI and the docked model of Mj0882. M.TaqI is shown in yellow, Mj0882 is shown in magenta.
Mentions: Docking of the target guanine to the Mj0882 structure (Figure 4) was guided by superposition of the "motif IV loop" of Mj0882 and M.TaqI, the only N-MTase co-crystallized with the nucleic acid substrate [29]. Under the assumption that both enzymes bind their targets in the same plane, the N2 group of guanine could be aligned with the N6 group of the adenine amino only if the purine ring was rotated by 120 degrees (i.e. with the atoms C2 and N3 of guanine superimposed onto atoms C6 and N1 of adenine). According to AutoDock [27] the energy of the interactions between the Mj0882-AdoMet complex and the docked guanine is quite favorable and comparable with that calculated for the target adenine in the M.TaqI structure (1g38) (-6.14 kcal/mol and -7.59 kcal/mol, respectively). These values are much lower than the energy of interactions between the protein and the cofactor (see above), because the AdoMet-binding groove is very deep and hydrophobic, while the base-binding site is relatively shallow. An alternative orientation of the target guanine in complex with 1dus could be obtained if its atoms C2 and N1 were superimposed onto atoms C6 and N1 of adenine. However, the latter model resulted in steric clashes between the ribose moiety and I132 (data not shown). Even if the flexible ligand docking option of AutoDock was used, which alleviated steric clashes, energies for the guanine in the "alternative" position (a cluster of similar conformations) were significantly worse (data not shown). In the first docked model the guanine moiety fits the groove on the protein's surface surprisingly well; a residue was identified, whose side chain might coordinate the ribose hydroxyl groups, namely D41 (Figure 3). Analysis of the multiple sequence alignment (Figure 1) revealed that this residue is highly conserved in the Mj0882, RsmD, RsmC, and YbiN families, supporting its functional importance. In the "alternative" docked model, D41 is too remote from the target base to make direct contacts to the target nucleoside (data not shown). However, a substantial conformational change upon substrate binding cannot be excluded, and direct participation of D41 in catalysis as well as other modes of guanine binding can be envisaged. Identification of this conserved carboxylate as a potentially important residue will hopefully prompt site-directed mutagenesis experiments and experimental determination of its role in binding and/or catalysis.

Bottom Line: Based on the results of sequence analysis and structure prediction, the residues involved in cofactor binding, target recognition and catalysis were identified, and the mechanism of the guanine-N2 methyltransfer reaction was proposed.Using the known Mj0882 structure, a comprehensive analysis of sequence-structure-function relationships in the family of genuine and putative m2G MTases was performed.The results provide novel insight into the mechanism of m2G methylation and will serve as a platform for experimental analysis of numerous uncharacterized N-MTases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bioinformatics Laboratory, International Institute of Cell and Molecular Biology, ul, ks, Trojdena 4, 02-109 Warsaw, Poland. iamb@wp.pl

ABSTRACT

Background: Escherichia coli guanine-N2 (m2G) methyltransferases (MTases) RsmC and RsmD modify nucleosides G1207 and G966 of 16S rRNA. They possess a common MTase domain in the C-terminus and a variable region in the N-terminus. Their C-terminal domain is related to the YbiN family of hypothetical MTases, but nothing is known about the structure or function of the N-terminal domain.

Results: Using a combination of sequence database searches and fold recognition methods it has been demonstrated that the N-termini of RsmC and RsmD are related to each other and that they represent a "degenerated" version of the C-terminal MTase domain. Novel members of the YbiN family from Archaea and Eukaryota were also indentified. It is inferred that YbiN and both domains of RsmC and RsmD are closely related to a family of putative MTases from Gram-positive bacteria and Archaea, typified by the Mj0882 protein from M. jannaschii (1dus in PDB). Based on the results of sequence analysis and structure prediction, the residues involved in cofactor binding, target recognition and catalysis were identified, and the mechanism of the guanine-N2 methyltransfer reaction was proposed.

Conclusions: Using the known Mj0882 structure, a comprehensive analysis of sequence-structure-function relationships in the family of genuine and putative m2G MTases was performed. The results provide novel insight into the mechanism of m2G methylation and will serve as a platform for experimental analysis of numerous uncharacterized N-MTases.

Show MeSH
Related in: MedlinePlus