Regulation of BRCA1 stability through the tandem UBX domains of isoleucyl-tRNA synthetase 1.

Aminoacyl-tRNA synthetases (ARSs) have evolved to acquire various additional domains. These domains allow ARSs to communicate with other cellular proteins in order to promote non-translational functions. Vertebrate cytoplasmic isoleucyl-tRNA synthetases (IARS1s) have an uncharacterized unique domain, UNE-I. Here, we present the crystal structure of the chicken IARS1 UNE-I complexed with glutamyl-tRNA synthetase 1 (EARS1). UNE-I consists of tandem ubiquitin regulatory X (UBX) domains that interact with a distinct hairpin loop on EARS1 and protect its neighboring proteins in the multi-synthetase complex from degradation. Phosphomimetic mutation of the two serine residues in the hairpin loop releases IARS1 from the complex. IARS1 interacts with BRCA1 in the nucleus, regulates its stability by inhibiting ubiquitylation via the UBX domains, and controls DNA repair function.

Structural Basis for the Antibiotic Resistance of Eukaryotic Isoleucyl-tRNA Synthetase.

Pathogenic aminoacyl-tRNA synthetases (ARSs) are attractive targets for anti-infective agents because their catalytic active sites are different from those of human ARSs. Mupirocin is a topical antibiotic that specifically inhibits bacterial isoleucy-ltRNA synthetase (IleRS), resulting in a block to protein synthesis. Previous studies on Thermus thermophilus IleRS indicated that mupirocin-resistance of eukaryotic IleRS is primarily due to differences in two amino acids, His581 and Leu583, in the active site. However, without a eukaryotic IleRS structure, the structural basis for mupirocin-resistance of eukaryotic IleRS remains elusive. Herein, we determined the crystal structure of Candida albicans IleRS complexed with Ile-AMP at 2.9 Å resolution. The largest difference between eukaryotic and prokaryotic IleRS enzymes is closure of the active site pocket by Phe55 in the HIGH loop; Arg410 in the CP core loop; and the second Lys in the KMSKR loop. The Ile-AMP product is lodged in a closed active site, which may restrict its release and thereby enhance catalytic efficiency. The compact active site also prevents the optimal positioning of the 9-hydroxynonanoic acid of mupirocin and plays a critical role in resistance of eukaryotic IleRS to anti-infective agents.

Dynamic assembly of Hda and the sliding clamp in the regulation of replication licensing.

Regulatory inactivation of DnaA (RIDA) is one of the major regulatory mechanisms of prokaryotic replication licensing. In RIDA, the Hda-sliding clamp complex loaded onto DNA directly interacts with adenosine triphosphate (ATP)-bound DnaA and stimulates the hydrolysis of ATP to inactivate DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. Here, we determined the crystal structure of the Hda-ß clamp complex. This complex contains two pairs of Hda dimers sandwiched between two ß clamp rings to form an octamer that is stabilized by three discrete interfaces. Two separate surfaces of Hda make contact with the ß clamp, which is essential for Hda function in RIDA. The third interface between Hda monomers occludes the active site arginine finger, blocking its access to DnaA. Taken together, our structural and mutational analyses of the Hda-ß clamp complex indicate that the interaction of the ß clamp with Hda controls the ability of Hda to interact with DnaA. In the octameric Hda-ß clamp complex, the inability of Hda to interact with DnaA is a novel mechanism that may regulate Hda function.