Eukaryotic lysyl-tRNA synthetases (LysRS) have an N-terminal appended tRNA-interaction domain (RID) that is absent in their prokaryotic counterparts. This domain is intrinsically disordered and lacks stable structures. The disorder-to-order transition is induced by tRNA binding and has implications on folding and subsequent assembly into multi-tRNA synthetase complexes. Here, we expressed and purified RID from human LysRS (hRID) in Escherichia coli and performed a detailed mutagenesis of the appended domain. hRID was co-purified with nucleic acids during Ni-affinity purification, and cumulative mutations on critical amino acid residues abolished RNA binding. Furthermore, we identified a structural ensemble between disordered and helical structures in non-RNA-binding mutants and an equilibrium shift for wild-type into the helical conformation upon RNA binding. Since mutations that disrupted RNA binding led to an increase in non-functional soluble aggregates, a stabilized RNA-mediated structural transition of the N-terminal appended domain may have implications on the functional organization of human LysRS and multi-tRNA synthetase complexes in vivo.
Bibliographical noteFunding Information:
Funding: This study was supported by grants from the National Research Foundation of Korea (NRF2015R1D1A4A01016640 and 2018M3A9H4079358) and the Ministry of Agriculture, Food and Rural Affairs (MAFRA; 716002-7) of the Korean Government.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Computer Science Applications
- Physical and Theoretical Chemistry
- Organic Chemistry
- Inorganic Chemistry