Crucial role of the C-terminal domain of Mycobacterium tuberculosis leucyl-tRNA synthetase in aminoacylation and editing

Abstract

The C-terminal extension of prokaryotic leucyl-tRNA synthetase (LeuRS) has been shown to make contacts with the tertiary structure base pairs of tRNALeu as well as its long variable arm. However, the precise role of the flexibly linked LeuRS C-terminal domain (CTD) in aminoacylation and editing processes has not been clarified. In this study, we carried out aspartic acid scanning within the CTD of Mycobacterium tuberculosis LeuRS (MtbLeuRS) and studied the effects on tRNALeu-binding capacity and enzymatic activity. Several critical residues were identified to impact upon the interactions between LeuRS and tRNALeu due to their contributions in the maintenance of structural stability or a neutral interaction interface between the CTD platform and tRNALeu elbow region. Moreover, we propose Arg921 as a crucial recognition site for the tRNALeu long variable arm in aminoacylation and tRNA-dependent pre-transfer editing. We also show here the CTD flexibility conferred by Val910 in regulation of LeuRS–tRNALeu interaction. Taken together, our results suggest the structural importance of the CTD in modulating precise interactions between LeuRS and tRNALeu during the quality control of leucyl-tRNALeu synthesis. This system for the investigation of the interactions between MtbLeuRS and tRNALeu provides a platform for the development of novel antitubercular drugs.

Identification of Lethal Mutations in Yeast Threonyl-tRNA Synthetase Revealing Critical Residues in Its Human Homolog

“…Binding of the Anticodon Controls tRNA Aminoacylation Activity-The C-terminal residues of aaRSs are sometimes essential for the enzymatic activity (48,49). In E. coli tRNA Thr , all of the bases from 32 to 37 interact through at least one hydrogen bond to the protein, and bases 35-38 are splayed out from the anticodon loop (14), indicating that the tRNA recognition cleft is a mobile region.…”

C-terminal Domain of Leucyl-tRNA Synthetase from Pathogenic Candida albicans Recognizes both tRNASer and tRNALeu

“…Although several conserved, positively charged residues were identified within the CTD of EcLeuRS, none of them provided site-specific interaction with tRNA Leu (28). (10,27). In addition to the three key lysine residues, the C-terminal five residues of CaLeuRS may maintain the proper conformation of the CTD during leucylation but not interact with tRNA Leu directly, which contrasts with archaeal PhLeuRS (10).…”

“…In EcLeuRS, Ala or Asp mutations of several conserved residues had only minimal effects on the aminoacylation activity as did the corresponding double and triple sites mutants (28). In another bacterial LeuRS, Mycobacterium tuberculosis LeuRS (MtLeuRS), several residues were shown to maintain the hydrophobic environment to stabilize the conformation of its CTD and to orient the tRNA (27 Leu has been studied extensively by structural and biochemical methods, the potential role of the CTD of eukaryotic LeuRSs in the recognition of tRNA Leu has never been reported. Here, our results first showed that CTD of CaLeuRS (CaCTD) is indispensible for leucylating both CatRNA Ser (CAG) and CatRNA Leu (CatRNAs).…”

A bridge between the aminoacylation and editing domains of leucyl-tRNA synthetase is crucial for its synthetic activity

“…Bovine serum albumin was used as a negative control to reflect that there was no fluorescence response to tRNA. The k d values were calculated by "one special binding equation" according to fitting fluorescence intensity change data vs. tRNA concentration, using Graphpad Prism software (Hu et al 2013). …”