Two non-redundant fragments in the N-terminal peptide of human cytosolic methionyl-tRNA synthetase were indispensable for the multi-synthetase complex incorporation and enzyme activity.

In human cytoplasm, nine aminoacyl-tRNA synthetases (aaRSs) and three protein factors form a multi-synthetase complex (MSC). Human cytosolic methionyl-tRNA synthetase (hcMetRS) is a component of the MSC. Sequence alignment revealed that hcMetRS has an N-terminal extension of 267 amino acid residues. This extension can be divided into three sub-domains: GST-like, GN, and GC sub-domains. The effect of each sub-domain in the N-terminal extension of hcMetRS on enzymatic activity and incorporation into the MSC was studied. The results of cellular assay showed that the GST-like sub-domain was responsible for the incorporation of hcMetRS into the MSC. The entire N-terminal extension of hcMetRS is indispensable for the enzymatic activity. Deletion mutagenesis revealed that a seven-amino acid motif within the sub-domain GC was important for the activity of amino acid activation. A conserved proline residue within the seven-amino acid motif was crucial, while the other six residues were moderately important for the amino acid activation activity. Thus, the last 15 residues of previously defined N-terminal extension of hcMetRS was a part of the catalytic domain; whereas the first 252 residues of hcMetRS constitute the N-terminal extended domain of hcMetRS. The formerly defined N-terminal extension of hcMetRS possesses two functions of two different domains.