Structure of the Thiostrepton Resistance Methyltransferase·S-Adenosyl-l-methionine Complex and Its Interaction with Ribosomal RNA

Abstract

The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr)·S-adenosyl-l-methionine (AdoMet) complex was determined at 2.45-Å 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.

Binding Induced RNA Conformational Changes Control Substrate Recognition and Catalysis by the Thiostrepton Resistance Methyltransferase (Tsr)

“…Table 1). Two binding events were previously observed for Tsr and other SPOUT family members using electrophoretic mobility shift assays (EMSAs) and the shifted bands were attributed to dimeric and tetrameric complexes of enzyme with RNA (7,20,27). We performed EMSAs using a similar range of Tsr concentrations as the FP experiments and confirmed the presence of these same complexes and their formation consistent with the K D values determined by FP (Fig.…”

“…Tsr Purification and Mutagenesis-Tsr was expressed from plasmid pET28a-Tsr in E. coli BL21(DE3)-pLysS as described previously (20), and purified using Ni 2ϩ affinity, heparin affinity, and gel filtration chromatographies. Elution volume from the Superdex 200 10/300 gel filtration column (GE Healthcare) was calibrated using Gel Filtration Standards (Bio-Rad).…”

“…Methylation Assays-Methylation assays using illustra MicroSpin G-25 columns (GE Healthcare) to separate 3 H-labeled RNA and the remaining [ 3 H]AdoMet were performed as described previously (20) with the following modifications. Assays were performed in 200-l reactions in assay buffer supplemented with 10% glycerol.…”

“…Second, a model 29-nt RNA hairpin containing the target loop and its associated stem, but lacking the full tertiary structure, was more readily methylated than the full 58-nt domain. These observations suggest that there may be an energetic penalty paid by the enzyme to unfold the RNA tertiary structure prior to catalysis (20). We therefore sought to determine whether Tsr must alter the RNA structure as part of its substrate recognition mechanism.…”

“…Tsr uses the co-substrate AdoMet to methylate the 23 S rRNA, presumably prior to the assembly of the 50 S subunit as the L11 and proposed Tsr binding surfaces are overlapping. The crystal structure of the Tsr⅐AdoMet complex definitively classified the enzyme as a member of the SPOUT family of methyltransferases (19,20). As such, it is an obligate homodimer through interactions mediated by its carboxyl-terminal domain (CTD; Fig.…”