Structural studies of eukaryotic aminoacyl-tRNA synthetases: Fungal Isoleucyl-tRNA synthetase and Metazoan Glutamyl- and Isoleucyl-tRNA synthetase complex

Abstract

Aminoacyl-tRNA synthetases (ARSs) catalyze the addition of amino acids to their cognate tRNAs with high fidelity in the initial step of protein synthesis. This canonical function of ARSs is essential to protein synthesis and is responsible for carrying accurate genetic information in every living organism. Although the catalytic units of ARSs are relatively well conserved, ARSs have acquired a new sequence motif and appended domains, which have no apparent connection to their aminoacylation reactions, to expand their functional capacity. In contrast to prokaryotic ARSs that do not form a complex, higher eukaryotic ARSs form a large and multi-tRNA synthetase complex. In this thesis, I will present two crystal eukaryotic ARS structures, those of fungal isoleucyl-tRNA synthetase (IleRS) and metazoan glutamyl-tRNA synthetase (GluRS) and IleRS complex. These structures provide insight into the structural basis of the antibiotic resistance of eukaryotic IleRS and the molecular basis of the interplay between metazoan GluRS and IleRS. First, I determined the crystal structure of Candida albicans IleRS complexed with Ile-AMP at a 2.9 Å resolution. The largest difference between eukaryotic and prokaryotic IleRS enzymes is the closure of the active site pocket by Phe55 in the HIGH loop, by Arg410 in the CP core loop, and by the second Lys in the KMSKR loop. The Ile-AMP product is lodged in a closed active site, which may restrict its release, thereby enhancing its catalytic efficiency. The compact active site also prevents the positioning of the 9-hydroxynonanoic acid in mupirocin and plays a critical role in the resistance of eukaryotic IleRS to anti-infective agents. Second, I determined the crystal structure of the Gallus gallus GluRS and IleRS complexes at a 2.4 Å resolution using serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs). Metazoan IleRSs have uncharacterized unique domains (UNE-Is), containing two repeat ~90 amino acid residues at the C-terminal end. UNE-I domains are composed of three repeat β-grasp folds, with each repeat containing one or two helices packed against four or five stranded antiparallel β–sheets. The second and third β-grasp domains of the UNE-I of the metazoan IleRS can be anchored to a distinct hairpin loop on the metazoan GluRS