Structural Insights into the Methylation of C1402 in 16S rRNA by Methyltransferase RsmI.

Mohan Zhao,Yuhui Dong,Linbo Zhang,Guangfeng Liu,Zengqiang Gao,Li Wang,Yong Gong,Jian Wang,Heng Zhang

doi:10.1371/journal.pone.0163816

Abstract

RsmI and RsmH are conserved S-Adenosylmethionine (AdoMet)-dependent methyltransferases (MTases) that are responsible for the 2′-O-methylation and N4-methylation of C1402 in bacterial 16S rRNA, respectively. Methylation of m4Cm1402 plays a role in fine-tuning the shape and functions of the P-site to increase the decoding fidelity, and was recently found to contribute to the virulence of Staphylococcus aureus in host animals. Here we report the 2.20-Å crystal structure of homodimeric RsmI from Escherichia coli in complex with the cofactor AdoMet. RsmI consists of an N-terminal putative RNA-binding domain (NTD) and a C-terminal catalytic domain (CTD) with a Rossmann-like fold, and belongs to the class III MTase family. AdoMet is specifically bound into a negatively charged deep pocket formed by both domains by making extensive contacts. Structure-based mutagenesis and isothermal titration calorimetry (ITC) assays revealed Asp100 and Ala124 are vital for AdoMet-binding. Although the overall fold of RsmI shows remarkable similarities to the characterized MTases involved in vitamin B12 biosynthesis, it exhibits a distinct charge distribution especially around the AdoMet-binding pocket because of different substrate specificity. The docking model of RsmI-AdoMet-RNA ternary complex suggested a possible base-flipping mechanism of the substrate RNA that has been observed in several known RNA MTases. Our structural and biochemical studies provide novel insights into the catalytic mechanism of C1402 methylation in 16S rRNA.

Highlights

Methylation of ribosomal RNA by methyltransferases (MTases) is closely associated with the fine-toned protein synthesis in ribosome and related physiological processes, such as 30S subunit assembly, fine-tuning of local rRNA structure and antibiotic resistance in somePLOS ONE | DOI:10.1371/journal.pone.0163816 October 6, 2016Structure-Function of 16S rRNA MTase RsmI
The structure of RsmI monomer is composed of two independent domains: the N-terminal domain (NTD) and the C-terminal domain (CTD) (Fig 1A and 1C)
C1402 modified by RsmI/RsmH is very close to A1408, both of which are located in the decoding center and require the mature 30S subunit as the substrate

Summary

Introduction

Methylation of ribosomal RNA (rRNA) by methyltransferases (MTases) is closely associated with the fine-toned protein synthesis in ribosome and related physiological processes, such as 30S subunit assembly, fine-tuning of local rRNA structure and antibiotic resistance in some. The rsmI and rsmH genes are conserved in most species of bacteria, and their homologs are discovered in several species of eukaryotes, suggesting that this modification at the Psite is a common structural feature of bacterial 16S rRNA [9]. Both single and double knockout strains of ΔrsmI/ΔrsmH can cause growth reduction compared with the wild-type in Escherichia coli [9], as observed in the effect of several point mutations in the P-site previously [11, 12]. A docking model of RsmI-AdoMet-RNA ternary complex is proposed to guide future investigations on the process of C1402 dimethylation in 16S rRNA

Results

Discussion

Conclusion

Materials and Methods