Structural basis for the methylation of G1405 in 16S rRNA by aminoglycoside resistance methyltransferase Sgm from an antibiotic producer: a diversity of active sites in m7G methyltransferases

Abstract

Sgm (Sisomicin-gentamicin methyltransferase) from antibiotic-producing bacterium Micromonospora zionensis is an enzyme that confers resistance to aminoglycosides like gentamicin and sisomicin by specifically methylating G1405 in bacterial 16S rRNA. Sgm belongs to the aminoglycoside resistance methyltransferase (Arm) family of enzymes that have been recently found to spread by horizontal gene transfer among disease-causing bacteria. Structural characterization of Arm enzymes is the key to understand their mechanism of action and to develop inhibitors that would block their activity. Here we report the structure of Sgm in complex with cofactors S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) at 2.0 and 2.1 A resolution, respectively, and results of mutagenesis and rRNA footprinting, and protein-substrate docking. We propose the mechanism of methylation of G1405 by Sgm and compare it with other m(7)G methyltransferases, revealing a surprising diversity of active sites and binding modes for the same basic reaction of RNA modification. This analysis can serve as a stepping stone towards developing drugs that would specifically block the activity of Arm methyltransferases and thereby re-sensitize pathogenic bacteria to aminoglycoside antibiotics.

Figure 1.

Structure of Sgm. Ribbon diagram of the Sgm–cofactor complex monomer. The N and C termini are labeled. The NTD and CTD domains of Sgm are colored in red and blue respectively with the core secondary structures labeled. The cofactor AdoMet is depicted in green. This figure and the following figures of this manuscript are prepared by PyMol (44).

Figure 2.

Stereo view of the final 2Fo-Fc electron density maps in the active-site region of Sgm. (A) Sgm–AdoMet complex. (B) Sgm–AdoHcy complex. These maps are contoured at a level of 1σ. (C) Stereo view of the superposition of Sgm–AdoMet and Sgm–AdoHcy complexes. AdoHcy is shown in orange and AdoMet in green respectively.

Figure 3.

ITC analyses. (A) Sgm–AdoHcy titration. (B) Sgm mutant R108A-AdoMet titration. (C) Sgm–GMP titration. The upper panels show the injection profile after baseline correction and the bottom panels show the integration (heat release) for each injection (except the first one).

Figure 4.

Schematic presentation of the Sgm–30S ribosomal subunit complex. (A) Summary of the footprinting results. Sections of the 16S rRNA that were analyzed by primer extension are shaded in grey. Nucleotides protected upon Sgm binding are indicated with a black dot, whereas exposed nucleotides are indicated with a black triangle symbol. For circle indicated regions, structural change was observed upon Sgm binding to 30S subunits. Helix numbering is presented in boxes. After chemical footprint, rRNA was extended with reverse transcriptase with primers complementary to regions: (1) 817–833, (2) 939–955 and (3) 1459–1479. (B) Mapping of footprinting data onto the 30S structure. Components of the 30S subunit are shown in the surface representation, ribosomal proteins are indicated in dark gray, 16S rRNA is indicated in light gray with the exception of helix 44 (residues 1400–1499), shown in white. Results of the footprinting experiments are color coded as follows: residues relatively more reactive in the footprinting experiment are shown in green, residues less reactive in the footprinting experiment are shown in red. The target G1405 is shown in yellow and is additionally indicated by a yellow circle. (C) Extreme steric clashes between Sgm and the 30S subunit occur, if the enzyme is docked to its target in helix 44 without any conformational changes. The 30S structure is color-coded as in panel B, but semi-transparent. Sgm is shown as a solid ribbon in blue; target G1405 is shown in yellow and helix 44 is shown in white.

Figure 5.

Model of guanosine 3′5′-bismonophosphate docked to the Sgm–AdoMet complex. Residues 1–63 have been omitted for clarity. The protein backbone is shown as a ribbon (with helices in violet and strands in yellow). Residues predicted to interact with the target nucleoside are shown as sticks and labeled; contacts predicted in detail are shown as green broken lines. A contact between the transferrable methyl group and the target N⁷ atom (distance = 3 Å) is shown by a red arrow.

Figure 6.

Orientation of AdoMet and guanosine in m⁷G MTases Ecm1 (position of AdoMet and guanosine determined experimentally), Sgm (docked guanosine), TrmB (docked AdoMet and guanosine) and RsmG (docked guanosine). Both ligand and the substrate have been presented in wireframe representation. Green structures correspond to positions occupied in Sgm, cyan in TrmB, magenta in RsmG and grey in Ecm1. On the left panel (A) the methyl group of AdoMet is indicated by a hashed circle, while on the right panel (B) it is not shown explicitly, instead its position is indicated by a red cross. The target N⁷ atom of guanosine in different complexes is indicated by a semi-transparent circle in the same color as the nucleoside.