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 Fasciola gigantica 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.
The cover image shows an artistic representation of the fluorescence-based assay of deadenylase activity used by Pavanello et al. in this issue, with fluorescence proportional to enzyme activity. Fluorescence (λem = 528 nm) was detected in a multi-mode reader using 96-well plates. Activity of different enzyme complexes (left to right) was measured at various time points (top to bottom). Controls (inactive enzyme preparations and no enzyme controls) were also included (bottom two rows). Pavanello et al. report that the central region of CNOT1 and CNOT9 stimulates deadenylation by the Ccr4–Not nuclease module (see pages 3437–3450).
Structure-function studies of the asparaginyl-tRNA synthetase from Fasciola gigantica: understanding the role of catalytic and non-catalytic domains
Vijayakumar Rajendran, Rohit Shukla, Harish Shukla, Timir Tripathi; Structure-function studies of the asparaginyl-tRNA synthetase from Fasciola gigantica: understanding the role of catalytic and non-catalytic domains. Biochem J 15 November 2018; 475 (21): 3377–3391. doi: https://doi.org/10.1042/BCJ20180700
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