Expression, purification and crystallization of adenosine 1408 aminoglycoside-resistance rRNA methyltransferases for structural studies

Abstract

High-level resistance to a broad spectrum of aminoglycoside antibiotics can arise through either N7-methyl guanosine 1405 (m⁷G1405) or N1-methyl adenosine 1408 (m¹A1408) modifications at the drug binding site in the bacterial 30S ribosomal subunit decoding center. Two distinct families of 16S ribosomal RNA (rRNA) methyltransferases that incorporate these modifications were first identified in aminoglycoside-producing bacteria but were more recently identified in both human and animal pathogens. These resistance determinants thus pose a new threat to the usefulness of aminoglycosides as antibiotics, demanding urgent characterization of their structures and activities. Here, we describe approaches to cloning, heterologous expression in Escherichia coli, and purification of two A1408 rRNA methyltransferases: KamB from the aminoglycoside-producer Streptoalloteichus tenebrarius and NpmA identified in a clinical isolate of pathogenic E. coli ARS3. Antibiotic minimum inhibitory concentration (MIC) assays and in vitro analysis of KamB and NpmA using circular dichroism (CD) spectroscopy, S-adenosyl-l-methionine (SAM) binding by isothermal titration calorimetry and 30S subunit methylation assays showed both enzymes were soluble, folded and active. Finally, crystals of each enzyme complexed with SAM were obtained, including selenomethionine-derived KamB, that will facilitate high-resolution X-ray crystallographic analyses of these important bacterial antibiotic-resistance determinants.

FIGURE 1. SDS-PAGE analysis of KamB protein expression and purification

For induction of protein expression (‘Cells’), samples shown correspond to ‘uninduced’ (lane U) and ‘induced’ (lane I) BL21 (DE3) cells grown in LB and autoinduction medium, respectively (sample loading was matched for cell density measured by absorbance at 600 nm). Purification from soluble supernatant (lane S) is indicated by analysis of samples following Heparin affinity (lane 1) and subsequent gel filtration (lane 2) chromatographies. Lane M is protein molecular weight marker (sizes indicated on right).

FIGURE 2. Measurement of A1408 16S rRNA methyltransferase-SAM binding by isothermal titration calorimetry (ITC)

Representative titrations and the derived dissociation constants (K_d) for (A) KamB-SAM and (B) NpmA-SAM interaction. K_d values shown are the average of three independent experiments.

FIGURE 3. Analysis of A1408 16S rRNA methyltransferase activity using an in vitro 30S subunit methylation assay

(A) Enrichment of methyltransferase activity during protein purification monitored by incorporation of ³H into 30S subunits from [³H]-SAM after 10 minutes. (B) Time course assays using final purified KamB and NpmA methyltransferases.

FIGURE 4. Circular dichroism (CD) spectroscopic analysis of KamB and NpmA

Protein CD spectra were deconvoluted using the CDSSTR algorithm via Dichroweb to produce the indicated protein secondary structure contents (see Materials and Methods).