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Structure of the thiostrepton resistance methyltransferase.S-adenosyl-L-methionine complex and its interaction with ribosomal RNA.

Dunstan MS, Hang PC, Zelinskaya NV, Honek JF, Conn GL - J. Biol. Chem. (2009)

Bottom Line: The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr).S-adenosyl-L-methionine (AdoMet) complex was determined at 2.45-A resolution.In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates.Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments.

View Article: PubMed Central - PubMed

Affiliation: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.

ABSTRACT
The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr).S-adenosyl-L-methionine (AdoMet) complex was determined at 2.45-A resolution. Tsr is definitively confirmed as a Class IV methyltransferase of the SpoU family with an N-terminal "L30-like" putative target recognition domain. The structure and our in vitro analysis of the interaction of Tsr with its target domain from 23 S ribosomal RNA (rRNA) demonstrate that the active biological unit is a Tsr homodimer. In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates. Molecular docking experiments predict that Tsr.rRNA binding is dictated entirely by the sequence and structure of the rRNA hairpin containing the A1067 target nucleotide and is most likely driven primarily by large complementary electrostatic surfaces. One L30-like domain is predicted to bind the target loop and the other is near an internal loop more distant from the target site where a nucleotide change (U1061 to A) also decreases methylation by Tsr. Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments. We therefore propose that Tsr achieves its absolute target specificity using the N-terminal domains of each monomer in combination to recognize the two distinct structural elements of the target rRNA hairpin such that both Tsr subunits contribute directly to the positioning of the target nucleotide on the enzyme.

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Molecular modeling of Tsr-rRNA interactions. A, four orthogonal views around the vertical axis of the Tsr dimer with electrostatic surface potential indicated in red (negative) and blue (positive). Docked RNA is shown in the two orientations on the right only. B, stereo view schematic of the docked Tsr·rRNA complex. Regions encompassing the A1067 target loop (magenta) recognized by the non-catalytic Tsr and the internal loop (cyan) recognized by the catalytic Tsr, including Phe-88 (also see Fig. 4), are shown in dashed boxes.
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Figure 3: Molecular modeling of Tsr-rRNA interactions. A, four orthogonal views around the vertical axis of the Tsr dimer with electrostatic surface potential indicated in red (negative) and blue (positive). Docked RNA is shown in the two orientations on the right only. B, stereo view schematic of the docked Tsr·rRNA complex. Regions encompassing the A1067 target loop (magenta) recognized by the non-catalytic Tsr and the internal loop (cyan) recognized by the catalytic Tsr, including Phe-88 (also see Fig. 4), are shown in dashed boxes.

Mentions: The target site for Tsr lies within the structurally well characterized L11-binding domain of 23 S rRNA (42, 43), allowing us to conduct molecular docking experiments. We first examined the electrostatic potential of the protein dimer surface. One face of the protein has a large stripe of positive surface surrounding the cleft between the two Tsr protomers (Fig. 3), with the NTD of each positioned on either side. In contrast, the reverse side is predominantly negatively charged across the center and is therefore very unlikely to have significant affinity for RNA. Rigid body docking was performed beginning with the 58-nucleotide rRNA domain oriented to face the positively lined cleft of the Tsr dimer. Docking experiments were performed using both shape-only and shape-electrostatics correlations, and each produced an extremely similar final docked orientation as the clear top solution (Fig. 3). The interactions predicted between Tsr and the rRNA extend over extensive complimentary surfaces and are largely electrostatic in nature. However, the exposed nature of the target loop and unusual structure of the internal bulge within Helix A leave open the possibility of direct recognition of base edges.


Structure of the thiostrepton resistance methyltransferase.S-adenosyl-L-methionine complex and its interaction with ribosomal RNA.

Dunstan MS, Hang PC, Zelinskaya NV, Honek JF, Conn GL - J. Biol. Chem. (2009)

Molecular modeling of Tsr-rRNA interactions. A, four orthogonal views around the vertical axis of the Tsr dimer with electrostatic surface potential indicated in red (negative) and blue (positive). Docked RNA is shown in the two orientations on the right only. B, stereo view schematic of the docked Tsr·rRNA complex. Regions encompassing the A1067 target loop (magenta) recognized by the non-catalytic Tsr and the internal loop (cyan) recognized by the catalytic Tsr, including Phe-88 (also see Fig. 4), are shown in dashed boxes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719339&req=5

Figure 3: Molecular modeling of Tsr-rRNA interactions. A, four orthogonal views around the vertical axis of the Tsr dimer with electrostatic surface potential indicated in red (negative) and blue (positive). Docked RNA is shown in the two orientations on the right only. B, stereo view schematic of the docked Tsr·rRNA complex. Regions encompassing the A1067 target loop (magenta) recognized by the non-catalytic Tsr and the internal loop (cyan) recognized by the catalytic Tsr, including Phe-88 (also see Fig. 4), are shown in dashed boxes.
Mentions: The target site for Tsr lies within the structurally well characterized L11-binding domain of 23 S rRNA (42, 43), allowing us to conduct molecular docking experiments. We first examined the electrostatic potential of the protein dimer surface. One face of the protein has a large stripe of positive surface surrounding the cleft between the two Tsr protomers (Fig. 3), with the NTD of each positioned on either side. In contrast, the reverse side is predominantly negatively charged across the center and is therefore very unlikely to have significant affinity for RNA. Rigid body docking was performed beginning with the 58-nucleotide rRNA domain oriented to face the positively lined cleft of the Tsr dimer. Docking experiments were performed using both shape-only and shape-electrostatics correlations, and each produced an extremely similar final docked orientation as the clear top solution (Fig. 3). The interactions predicted between Tsr and the rRNA extend over extensive complimentary surfaces and are largely electrostatic in nature. However, the exposed nature of the target loop and unusual structure of the internal bulge within Helix A leave open the possibility of direct recognition of base edges.

Bottom Line: The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr).S-adenosyl-L-methionine (AdoMet) complex was determined at 2.45-A resolution.In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates.Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments.

View Article: PubMed Central - PubMed

Affiliation: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.

ABSTRACT
The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr).S-adenosyl-L-methionine (AdoMet) complex was determined at 2.45-A resolution. Tsr is definitively confirmed as a Class IV methyltransferase of the SpoU family with an N-terminal "L30-like" putative target recognition domain. The structure and our in vitro analysis of the interaction of Tsr with its target domain from 23 S ribosomal RNA (rRNA) demonstrate that the active biological unit is a Tsr homodimer. In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates. Molecular docking experiments predict that Tsr.rRNA binding is dictated entirely by the sequence and structure of the rRNA hairpin containing the A1067 target nucleotide and is most likely driven primarily by large complementary electrostatic surfaces. One L30-like domain is predicted to bind the target loop and the other is near an internal loop more distant from the target site where a nucleotide change (U1061 to A) also decreases methylation by Tsr. Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments. We therefore propose that Tsr achieves its absolute target specificity using the N-terminal domains of each monomer in combination to recognize the two distinct structural elements of the target rRNA hairpin such that both Tsr subunits contribute directly to the positioning of the target nucleotide on the enzyme.

Show MeSH
Related in: MedlinePlus