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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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Related in: MedlinePlus

Multiple alignment of the Mj0882, RsmC, RsmD, and YbiN families. Sequences are denoted by the species' name and the NCBI gene identification number; sequences lacking the number were obtained from the unfinished genome data. Numbers on the left side indicate the index of the first N-terminal residue shown, numbers in parentheses indicate how many residues in an insertion were omitted for clarity. Conserved motifs are labeled according to the nomenclature described for the AdoMet-dependent MTase superfamily [12]. Conserved residues are highlighted in black; the residues with invariant physicochemical character (hydrophobic, small etc.) are highlighted in gray. Conserved carboxylates predicted to bind AdoMet are shown in black on red background; the pair of residues proposed to make van der Waals contacts to guanine is shown in green on a yellow background; the Asn residue (His in Archaeal YbiN orthologs) proposed to catalyze the methyltransfer is shown in yellow on red background; the carboxylate proposed to bind the ribose of the target nucleoside is shown in yellow on magenta background. Secondary structure elements of Mj0882, extracted from the 1dus coordinates and virtually identical for the secondary structure independently predicted for other families (see Methods), are shown above the alignment.
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Figure 1: Multiple alignment of the Mj0882, RsmC, RsmD, and YbiN families. Sequences are denoted by the species' name and the NCBI gene identification number; sequences lacking the number were obtained from the unfinished genome data. Numbers on the left side indicate the index of the first N-terminal residue shown, numbers in parentheses indicate how many residues in an insertion were omitted for clarity. Conserved motifs are labeled according to the nomenclature described for the AdoMet-dependent MTase superfamily [12]. Conserved residues are highlighted in black; the residues with invariant physicochemical character (hydrophobic, small etc.) are highlighted in gray. Conserved carboxylates predicted to bind AdoMet are shown in black on red background; the pair of residues proposed to make van der Waals contacts to guanine is shown in green on a yellow background; the Asn residue (His in Archaeal YbiN orthologs) proposed to catalyze the methyltransfer is shown in yellow on red background; the carboxylate proposed to bind the ribose of the target nucleoside is shown in yellow on magenta background. Secondary structure elements of Mj0882, extracted from the 1dus coordinates and virtually identical for the secondary structure independently predicted for other families (see Methods), are shown above the alignment.

Mentions: The amino-acid sequences of E. coli RsmC and RsmD were used in PSI-BLAST database searches to identify orthologous proteins (hits reported in the 1st iteration with e-values <10-40 and sequence identity > 40%; see Materials and Methods for details). The resulting multiple sequence alignment (Figure 1) was used to predict the secondary structure (separately for both protein families) and to precisely identify boundaries of the C-terminal catalytic domain. The Rsm sequences were divided into the N-terminal (variable) and C-terminal (conserved) domains (hereafter dubbed NTD and CTD) of approximately equal length. The sequences of NTD and CTD of RsmC and RsmD were used in additional PSI-BLAST searches and also submitted to the structure prediction MetaServer http://bioinfo.pl. The CTD exhibited similarity to a large class of genuine and predicted N-methyltransferases. Both the RsmC and RsmD CTD sequences showed highest similarity to each other (e-values in the range of 10-20–10-19 in the 1st iteration), but also to a family of putative MTases from Gram-positive Bacteria and Archaea, typified by the Mj0882 protein from M. jannaschii (e-value 10-19 in the 2nd iteration starting with RsmC, 10-35 in the 3rd iteration starting with RsmD). Interestingly, the crystal structure of Mj0882 has been solved as a part of a structural genomics program (1dus; deposited in the Protein Data Bank in January 2000, cited as: LW Hung, L Huang, R Kim, SH Kim: Crystal structure and functional analysis of a hypothetical protein, Mj0882, from Methanococcus jannaschii, to be published). However, neither the biochemical characterization of this protein or the analysis of its structure has been published and its function remains a mystery. More distant homologs with shorter alignments reported in the CTD-initiated search included the HemK/YfcB family of protenr:N5-glutamine MTases [14,15] and various distinct RNA and DNA MTase families (e-values > 10-16 after the RsmD-initiated search converged in the 4th iteration). Only Mj0882 and its close homologs could be aligned to the CTD of RsmC or RsmD over their entire length if the default PSI-BLAST parameters were used. Reciprocal database searches initiated with Mj0882 yielded its presumed orthologs from Archaea and Gram-positive Bacteria (detected in the 1st PSI-BLAST iteration with e-values < 10-30 and sequence identity to the query > 40%) and CTD sequences of proteobacterial m2G MTases (RsmC detected in the 2nd iteration, e-values ≥ 2*10-26 and sequence identities ≤ 26%; RsmD detected in the 3rd iteration with e values ≥ 2*10-27 and sequence identities ≤ 24%). This result of reciprocal database searches strongly suggested an orthologous relationship [16] between the RsmC/RsmD and Mj0882 families. Further support for this prediction was obtained from the threading analysis. Among structurally characterized MTases, Mj0882 was reported as the definitely best structural template for the CTD of RsmC and RsmD (confident Pcons scores 5.78 and 6.09; see Table 1 and Table 2 for the summary of the threading analysis).


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)

Multiple alignment of the Mj0882, RsmC, RsmD, and YbiN families. Sequences are denoted by the species' name and the NCBI gene identification number; sequences lacking the number were obtained from the unfinished genome data. Numbers on the left side indicate the index of the first N-terminal residue shown, numbers in parentheses indicate how many residues in an insertion were omitted for clarity. Conserved motifs are labeled according to the nomenclature described for the AdoMet-dependent MTase superfamily [12]. Conserved residues are highlighted in black; the residues with invariant physicochemical character (hydrophobic, small etc.) are highlighted in gray. Conserved carboxylates predicted to bind AdoMet are shown in black on red background; the pair of residues proposed to make van der Waals contacts to guanine is shown in green on a yellow background; the Asn residue (His in Archaeal YbiN orthologs) proposed to catalyze the methyltransfer is shown in yellow on red background; the carboxylate proposed to bind the ribose of the target nucleoside is shown in yellow on magenta background. Secondary structure elements of Mj0882, extracted from the 1dus coordinates and virtually identical for the secondary structure independently predicted for other families (see Methods), are shown above the alignment.
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Related In: Results  -  Collection

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

Figure 1: Multiple alignment of the Mj0882, RsmC, RsmD, and YbiN families. Sequences are denoted by the species' name and the NCBI gene identification number; sequences lacking the number were obtained from the unfinished genome data. Numbers on the left side indicate the index of the first N-terminal residue shown, numbers in parentheses indicate how many residues in an insertion were omitted for clarity. Conserved motifs are labeled according to the nomenclature described for the AdoMet-dependent MTase superfamily [12]. Conserved residues are highlighted in black; the residues with invariant physicochemical character (hydrophobic, small etc.) are highlighted in gray. Conserved carboxylates predicted to bind AdoMet are shown in black on red background; the pair of residues proposed to make van der Waals contacts to guanine is shown in green on a yellow background; the Asn residue (His in Archaeal YbiN orthologs) proposed to catalyze the methyltransfer is shown in yellow on red background; the carboxylate proposed to bind the ribose of the target nucleoside is shown in yellow on magenta background. Secondary structure elements of Mj0882, extracted from the 1dus coordinates and virtually identical for the secondary structure independently predicted for other families (see Methods), are shown above the alignment.
Mentions: The amino-acid sequences of E. coli RsmC and RsmD were used in PSI-BLAST database searches to identify orthologous proteins (hits reported in the 1st iteration with e-values <10-40 and sequence identity > 40%; see Materials and Methods for details). The resulting multiple sequence alignment (Figure 1) was used to predict the secondary structure (separately for both protein families) and to precisely identify boundaries of the C-terminal catalytic domain. The Rsm sequences were divided into the N-terminal (variable) and C-terminal (conserved) domains (hereafter dubbed NTD and CTD) of approximately equal length. The sequences of NTD and CTD of RsmC and RsmD were used in additional PSI-BLAST searches and also submitted to the structure prediction MetaServer http://bioinfo.pl. The CTD exhibited similarity to a large class of genuine and predicted N-methyltransferases. Both the RsmC and RsmD CTD sequences showed highest similarity to each other (e-values in the range of 10-20–10-19 in the 1st iteration), but also to a family of putative MTases from Gram-positive Bacteria and Archaea, typified by the Mj0882 protein from M. jannaschii (e-value 10-19 in the 2nd iteration starting with RsmC, 10-35 in the 3rd iteration starting with RsmD). Interestingly, the crystal structure of Mj0882 has been solved as a part of a structural genomics program (1dus; deposited in the Protein Data Bank in January 2000, cited as: LW Hung, L Huang, R Kim, SH Kim: Crystal structure and functional analysis of a hypothetical protein, Mj0882, from Methanococcus jannaschii, to be published). However, neither the biochemical characterization of this protein or the analysis of its structure has been published and its function remains a mystery. More distant homologs with shorter alignments reported in the CTD-initiated search included the HemK/YfcB family of protenr:N5-glutamine MTases [14,15] and various distinct RNA and DNA MTase families (e-values > 10-16 after the RsmD-initiated search converged in the 4th iteration). Only Mj0882 and its close homologs could be aligned to the CTD of RsmC or RsmD over their entire length if the default PSI-BLAST parameters were used. Reciprocal database searches initiated with Mj0882 yielded its presumed orthologs from Archaea and Gram-positive Bacteria (detected in the 1st PSI-BLAST iteration with e-values < 10-30 and sequence identity to the query > 40%) and CTD sequences of proteobacterial m2G MTases (RsmC detected in the 2nd iteration, e-values ≥ 2*10-26 and sequence identities ≤ 26%; RsmD detected in the 3rd iteration with e values ≥ 2*10-27 and sequence identities ≤ 24%). This result of reciprocal database searches strongly suggested an orthologous relationship [16] between the RsmC/RsmD and Mj0882 families. Further support for this prediction was obtained from the threading analysis. Among structurally characterized MTases, Mj0882 was reported as the definitely best structural template for the CTD of RsmC and RsmD (confident Pcons scores 5.78 and 6.09; see Table 1 and Table 2 for the summary of the threading analysis).

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