Structure-function studies of the asparaginyl-tRNA synthetase from understanding the role of catalytic and non-catalytic domains.

The asparaginyl-tRNA synthetase (NRS) catalyzes the attachment of asparagine to its cognate tRNA during translation. NRS first catalyzes the binding of Asn and ATP to form the NRS-asparaginyl adenylate complex, followed by the esterification of Asn to its tRNA. We investigated the role of constituent domains in regulating the structure and activity of NRS (FgNRS). We cloned the full-length FgNRS, along with its various truncated forms, expressed, and purified the corresponding proteins. Size exclusion chromatography indicated a role of the anticodon-binding domain (ABD) of FgNRS in protein dimerization. The N-terminal domain (NTD) was not essential for cognate tRNA binding, and the hinge region between the ABD and the C-terminal domain (CTD) was crucial for regulating the enzymatic activity. Molecular docking and fluorescence quenching experiments elucidated the binding affinities of the substrates to various domains. The molecular dynamics simulation of the modeled protein showed the presence of an unstructured region between the NTD and ABD that exhibited a large number of conformations over time, and further analysis indicated this region to be intrinsically disordered. The present study provides information on the structural and functional regulation, protein-substrate(s) interactions and dynamics, and the role of non-catalytic domains in regulating the activity of FgNRS.