Structural Basis for the Recognition of Isoleucyl-Adenylate and an Antibiotic, Mupirocin, by Isoleucyl-tRNA Synthetase

Abstract

An analogue of isoleucyl-adenylate (Ile-AMS) potently inhibits the isoleucyl-tRNA synthetases (IleRSs) from the three primary kingdoms, whereas the antibiotic mupirocin inhibits only the eubacterial and archaeal IleRSs, but not the eukaryotic enzymes, and therefore is clinically used against methicillin-resistant Staphylococcus aureus. We determined the crystal structures of the IleRS from the thermophilic eubacterium, Thermus thermophilus, in complexes with Ile-AMS and mupirocin at 3.0-and 2.5-Å resolutions, respectively. A structural comparison of the IleRS⅐Ile-AMS complex with the adenylate complexes of other aminoacyl-tRNA synthetases revealed the common recognition mode of aminoacyladenylate by the class I aminoacyl-tRNA synthetases. The Ile-AMS and mupirocin, which have significantly different chemical structures, are recognized by many of the same amino acid residues of the IleRS, suggesting that the antibiotic inhibits the enzymatic activity by blocking the binding site of the high energy intermediate, Ile-AMP. In contrast, the two amino acid residues that concomitantly recognize Ile-AMS and mupirocin are different between the eubacterial/archaeal IleRSs and the eukaryotic IleRSs. Mutagenic analyses revealed that the replacement of the two residues significantly changed the sensitivity to mupirocin. Aminoacyl-tRNA synthetases (aaRSs)1 esterify the cognate amino acids with their specific tRNAs via the following twostep reactions: the formation of aminoacyl-adenylate (aa-AMP), an active intermediate, from an amino acid and ATP; and the transfer of the aminoacyl moiety to the 3Ј-terminal adenosine (A 76 ) of tRNA. The aaRSs can be divided into two classes, class I and class II, comprising 10 members each, which have distinct catalytic domain architectures with exclusive signature motifs for the ATP binding (1). The class I aaRSs have a catalytic domain constructed with the Rossmann fold, and display two signature amino acid motifs "HIGH" and "KMSKS." The Rossmann fold has a ␤ 6 ␣ 4 topology and is apparently divided into two symmetrical halves (␤ 3 ␣ 2 topology each), which are believed to have evolved by a genetic event such as gene duplication. The class I aaRSs are further divided into three subclasses (class Ia, Ib, and Ic) on the basis of the sequence homology and the domain architectures (2). The class Ia aaRSs consist of the isoleucyl-, methionyl-, valyl-, leucyl-, cysteinyl-, and arginyl-tRNA synthetases (IleRS, MetRS, ValRS, LeuRS, CysRS, and ArgRS, respectively); the class Ib aaRSs include the glutamyl-and glutaminyl-tRNA synthetases GluRS and GlnRS, respectively, and the class Ic aaRSs are the tyrosyl-and tryptophanyl-tRNA synthetases (TyrRS and TrpRS, respectively).In general, an aa-AMP analogue potently inhibits the corresponding aaRSs from eubacteria, archaea, and eukaryotes. In contrast, some antibiotics inhibit only the eubacterial (and archaeal) aaRSs, but not the eukaryotic enzymes. The best known example is mupirocin, which targets bacterial and archaeal IleRSs (6, 7). Mupirocin (the che...

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