Molecular basis of the multifaceted functions of human leucyl-tRNA synthetase in protein synthesis and beyond.

Andrés Palencia,En-Duo Wang,Hao Li,Jinzhong Lin,Jing Li,Jinghua Zhao,Mingzhu Wang,Ru-Juan Liu,Stephen Cusack,Tao Long

doi:10.1093/nar/gkaa189

Abstract

Human cytosolic leucyl-tRNA synthetase (hcLRS) is an essential and multifunctional enzyme. Its canonical function is to catalyze the covalent ligation of leucine to tRNALeu, and it may also hydrolyze mischarged tRNAs through an editing mechanism. Together with eight other aminoacyl-tRNA synthetases (AaRSs) and three auxiliary proteins, it forms a large multi-synthetase complex (MSC). Beyond its role in translation, hcLRS has an important moonlight function as a leucine sensor in the rapamycin complex 1 (mTORC1) pathway. Since this pathway is active in cancer development, hcLRS is a potential target for anti-tumor drug development. Moreover, LRS from pathogenic microbes are proven drug targets for developing antibiotics, which however should not inhibit hcLRS. Here we present the crystal structure of hcLRS at a 2.5 Å resolution, the first complete structure of a eukaryotic LRS, and analyze the binding of various compounds that target different sites of hcLRS. We also deduce the assembly mechanism of hcLRS into the MSC through reconstitution of the entire mega complex in vitro. Overall, our study provides the molecular basis for understanding both the multifaceted functions of hcLRS and for drug development targeting these functions.

Highlights

Aminoacyl-tRNA synthetases (AaRSs) catalyze the attachment of amino acids (AAs) to their cognate transfer RNAs to provide aminoacyl-tRNAs for translation
Full length Human cytosolic leucyl-tRNA synthetase (hcLRS) (FL-hcLRS, Supplementary Figure S1) which contains 1176 amino acids (AA) could be crystallized but the crystals only diffracted to 10 Aresolution
Our results suggest that the binding of tRNA and RagD by hcLRS are via different regions from two discrete domains of the vertebrate C-terminal (VC) domain

Summary

Introduction

Aminoacyl-tRNA synthetases (AaRSs) catalyze the attachment of amino acids (AAs) to their cognate transfer RNAs (tRNAs) to provide aminoacyl-tRNAs for translation. This process of aminoacylation occurs in two steps: (i) the formation of the activated aminoacyl-adenylate intermediate (aaAMP) and (ii) the transfer of the aminacyl moiety to the 3 -end of the tRNA [1,2]. Some non-cognate AAs that are structurally similar to the cognate one, can be mis-activated or mis-charged to the tRNA by AaRSs [3]. To correct mistakes of this kind, some AaRSs have evolved a proofreading (editing) activity to hydrolyze mis-activated AAs (pretransfer editing) or mis-charged AA-tRNA (post-transfer editing) [4,5]. Increasing evidence shows that these nonclassical functions of eukaryotic AaRSs are normally endowed by additional domains and motifs appended during evolution, which make eukaryotic AaRSs multifunctional, and their structural study important [9–12]

Methods

Results

Conclusion