Recombinant Legionella pneumophila subsp. pneumophila Ribosomal RNA large subunit methyltransferase N (rlmN)

What experimental approaches are used to determine the structure and function of recombinant Legionella pneumophila rlmN?

To characterize rlmN, researchers employ X-ray crystallography to resolve atomic-level structures and ITC to quantify ligand-binding affinities. For example, the apo and S-adenosyl-L-methionine (AdoMet)-bound structures of Lpg2936 (a homolog) were solved at 1.5 Å and 2.3 Å resolution, respectively, revealing a conserved SPOUT methyltransferase fold with a trefoil knot topology . The dimerization interface, critical for enzymatic activity, spans 1,480 Ų and involves 28 hydrogen bonds and 15 salt bridges . ITC experiments demonstrated asymmetric two-site AdoMet binding, with dissociation constants (Kd) varying between 1–10 μM, suggesting cooperative ligand interactions .

Key methodological considerations:

• Crystallization conditions: Optimize pH (6.0–7.5) and polyethylene glycol (PEG) concentrations to stabilize the SPOUT domain.

• Ligand-binding assays: Use ITC to validate stoichiometry and affinity, accounting for cooperative effects.

How does rlmN’s catalytic mechanism differ from other RNA methyltransferases?

rlmN belongs to the SPOUT methyltransferase family, which utilizes a unique trefoil knot structure to bind AdoMet and catalyze methyl group transfer to rRNA. Unlike Rossmann-fold methyltransferases, SPOUT enzymes like rlmN require dimerization for activity. Structural comparisons show that residues Arg222 and Lys100 in L. pneumophila Lpg2936 stabilize AdoMet binding through salt bridges, while the PUA domain positions the RNA substrate . Mutagenesis of these residues reduces methylation efficiency by >90%, confirming their functional necessity .

Contradiction analysis: While most SPOUT methyltransferases target 16S rRNA (e.g., RsmE modifies U1498 in E. coli), rlmN homologs in Legionella may exhibit divergent substrate specificity. Resolve this by conducting cross-species complementation assays or ribosome profiling after gene knockout.

What role does rlmN play in Legionella pneumophila pathogenesis?

Although direct evidence linking rlmN to virulence remains limited, studies on L. pneumophila’s RNA-level immune evasion suggest methyltransferases enhance ribosomal efficiency during infection. For instance, Legionella secretes small RNAs (e.g., RsmY) that mimic host microRNAs to dampen RIG-I-mediated immune responses . Efficient translation of virulence factors likely depends on rRNA modifications by enzymes like rlmN.

Experimental validation:

• Knockout strains: Compare the growth kinetics of ΔrlmN and wild-type L. pneumophila in macrophage infection models.

• Ribosome sequencing: Identify methylation sites in 23S rRNA under stress conditions (e.g., nutrient deprivation, host cell exposure).

How can structural data guide the design of rlmN inhibitors?

The AdoMet-binding pocket in rlmN homologs is a prime target for inhibitor development. In Lpg2936, AdoMet adopts a bent conformation stabilized by residues Tyr9 and Glu103 . Virtual screening of small molecules that mimic this conformation could yield competitive inhibitors.

Structural insights for drug design:

• Hotspot residues: Tyr9 (π-stacking with AdoMet’s adenine), Arg222 (hydrogen bonds to AdoMet’s carboxyl group).

• Dimer disruption: Compounds targeting the SPOUT dimer interface (e.g., helix α7) may allosterically inhibit activity.

What challenges arise when expressing recombinant rlmN in heterologous systems?

L. pneumophila rlmN contains disordered loops (e.g., β11/α7 loop) that complicate soluble expression. A study achieving 85% solubility used E. coli BL21(DE3) with a codon-optimized gene, 0.5 mM IPTG induction at 18°C, and a His-tag purification system .

Optimization workflow:

• Codon optimization: Adjust GC content to match the host’s tRNA pool.

• Induction conditions: Screen temperatures (16–25°C) and IPTG concentrations (0.1–1.0 mM).

• Buffer additives: Include 200 mM NaCl and 5% glycerol to stabilize the SPOUT domain.

How do post-translational modifications affect rlmN activity?

Phosphorylation at Ser56 and acetylation at Lys89 in homologous enzymes modulate RNA-binding affinity. In E. coli RsmE, phosphorylation reduces methyltransferase activity by 40%–60% . To assess this in Legionella:

• Mass spectrometry: Identify modification sites in recombinant rlmN.

• Kinase assays: Incubate rlmN with Legionella lysates and ATP, then measure methylation rates.

What discrepancies exist between in vitro and in vivo studies of rlmN function?

• CRISPR interference: Titrate rlmN expression levels to identify phenotypic thresholds.

• RNA immunoprecipitation: Verify rRNA targets in native ribosomes.

How can researchers validate the methyltransferase activity of recombinant rlmN?

Use radiolabeled AdoMet (³H-SAM) assays:

• Incubate 1 μM rlmN with 10 μM ³H-SAM and 5 μM rRNA substrate (30°C, pH 7.5).

• Quench reactions with 0.1% trifluoroacetic acid.

• Measure incorporated radioactivity via scintillation counting.

Controls:

• Omit rRNA to assess non-specific methylation.

• Use catalytically dead mutants (e.g., D146A) to confirm enzyme dependence.