Small methyltransferase RlmH assembles a composite active site to methylate a ribosomal pseudouridine

Abstract

Eubacterial ribosomal large-subunit methyltransferase H (RlmH) methylates 23S ribosomal RNA pseudouridine 1915 (Ψ1915), which lies near the ribosomal decoding center. The smallest member of the SPOUT superfamily of methyltransferases, RlmH lacks the RNA recognition domain found in larger methyltransferases. The catalytic mechanism of RlmH enzyme is unknown. Here, we describe the structures of RlmH bound to S-adenosyl-methionine (SAM) and the methyltransferase inhibitor sinefungin. Our structural and biochemical studies reveal catalytically essential residues in the dimer-mediated asymmetrical active site. One monomer provides the SAM-binding site, whereas the conserved C-terminal tail of the second monomer provides residues essential for catalysis. Our findings elucidate the mechanism by which a small protein dimer assembles a functionally asymmetric architecture.

Figure 1

Structure and dimer formation of RlmH. (A) Crystal structure of the RlmH · SAM homodimer (upper panel) and close-up view of the active site (lower panel). The apo protein (light gray, PDB ID 1NS5) is superimposed on the SAM-bound protein (dark gray). Sticks show active site residues (apo: light blue; SAM-bound: pink. (B) Three bights (bight 1: blue; bight 2: cyan; bight 3: orange) form the SAM-binding pocket of monomer A. Teal: unbiased F_obs-F_calc electron density; pink: monomer A; gray: monomer B; magenta: conserved C-terminal tail of monomer B. (C) Comparison of the conformations of the C-terminus in the apo- (blue) and SAM-bound protein (magenta and pink). (D) Molecular weights of RlmH and four mutants used in this study were determined by SEC/MALS (RI/UV). UV traces of proteins eluted from Superdex S-200 are shown. For clarity, only every tenth measurement of molar mass within the range of the elution volume is plotted. Two plots with different concentration of injected samples are shown. Gray arrows indicate the axes to which the graphs correspond. See also Figures S1, S2, S3 and S9 and Tables S1 and S2.

Figure 2

Kinetic analyses of wild-type and mutant RlmH. Initial velocities (V ₀, µM min⁻¹) of methylation of ΔrlmH−70S ribosome (0.3 µM) by wild-type or mutant RlmH at 25 °C with 50 µM [³H]-labeled SAM. Error bars show error of fit for two independent experiments.

Figure 3

ITC analyses of SAM binding to wild-type or mutant RlmH. (A) 50 µM RlmH wild-type; (B) 51 µM RlmH-H129A; (C) 50 µM RlmH-E138A; (D) 51 µM RlmH-E138Q; (E) 35 µM RlmH-Y152F; (F) 51 µM RlmH-H153F and (G) 29 µM RlmH-R154A. Dissociation constants (K _D) of SAM for each RlmH mutant are calculated from three replicates (except R154A, n = 2). Data were fitted using a sequential binding model. The c-value was 10. See also Figure S7.

Figure 4

Competition analyses of RlmH mutants. (A) Scheme for the in vitro competitive methylation assay. (B–F) Time progress curves for methylation of ΔrlmH-70S ribosomes by wild-type RlmH (1 µM) in competition with E138A, E138Q, Y152F, H153F and R154A mutants at different concentrations. The same curve for methylation by wild-type RlmH is shown (black) in all panels for reference. Error bars show error of fit for two independent experiments.

Figure 5

Comparison of the active sites of RlmH and other SPOUT methyltransferases (OrfX and TrmD), containing the conserved D/E-R-Y residues. (A) Predicted model of the 70S· RlmH · SAM complex (gray; Purta et al.) and SAM-bound RlmH (magenta, this work). SAM is shown in sticks (magenta). Predicted position of Ψ1915 is shown with sticks. (B) The active site of S. aureus rRNA methyltransferase OrfX (yellow) in complex with SAM (yellow, sticks) and a phosphate ion (orange) (PDB ID 4FAK). SAM is bound to one OrfX monomer, while the residues shown in sticks are provided by the second monomer, as in E. coli RlmH. Conserved (Asp/Glu), Arg and Tyr interacting with the substrate nucleotide/analogs are shown in sticks and labeled. (C) The active site of Haemophilus influenza TrmD (light blue/dark blue) in complex with sinefungin (SFG, cyan, sticks) and tRNA (yellow) (PDB ID 4YVI). (D) The active site of E. coli RlmH (magenta/pink) in complex with SAM (cyan, sticks), shown in a view similar to that for the active site of H. influenza TrmD in (C). The catalytic C-terminal tail is colored in red. (E) Schematic of the mechanism of Ψ1915 methylation by RlmH. See also Figures S5, S6 and S8.