Abstract
All seryl-tRNA synthetases (SerRSs) are functional homodimers with a C-terminal active site domain typical for class II aminoacyl-tRNA synthetases and an N-terminal domain involved in tRNA binding. The recently solved three-dimensional structure of Methanosarcina barkeri SerRS revealed the idiosyncratic features of methanogenic-type SerRSs; that is, an active site zinc ion, a unique tRNA binding domain, and an insertion of approximately 30 residues in the catalytic domain, which adopt a helix-turn-helix (HTH) fold. Here, we present biochemical evidence for multiple roles of the HTH motif; it is important for dimerization of the enzyme, contributes to the overall stability, and is critical for the proper positioning of the tRNA binding domain relative to the catalytic domain. The changes in intrinsic fluorescence during denaturation of the wild-type M. barkeri SerRS and of the mutated variant lacking the HTH motif combined with cross-linking and gel analysis of protein subunits during various stages of the unfolding process revealed significantly reduced stability of the mutant dimers. In vitro kinetic analysis of enzymes, mutated in one of the N-terminal helices and the HTH motif, shows impaired tRNA binding and aminoacylation and emphasizes the importance of this domain for the overall architecture of the enzyme. The role of the idiosyncratic HTH motif in dimer stabilization and association between the catalytic and tRNA binding domain has been additionally confirmed by a yeast two-hybrid approach. Furthermore, we provide experimental evidence that tRNA binds across the dimer.
Highlights
The fidelity of protein synthesis relies upon the interpretation of the genetic code by aminoacyl-tRNA synthetases.2 Each enzyme from this family catalyzes covalent
In tetrameric AlaRS, motif 1 is not involved in any intermolecular interactions, as the C-terminal domain is responsible for enzyme oligomerization [18]
Organizing Interactions between Synthetase Domains—With the addition of the tRNA binding domains to catalytic cores, which occurred during evolution via multiple gene fusions, modern synthetases gained the capacity to bind cognate tRNA molecules, improving the discrimination against non-cognate tRNA substrates in the cell [19]
Summary
The fidelity of protein synthesis relies upon the interpretation of the genetic code by aminoacyl-tRNA synthetases (aaRSs). Each enzyme from this family catalyzes covalent. In the bacterial-type SerRS, as revealed by studies on the synthetases of Escherichia coli and Thermus thermophilus, each subunit possesses a C-terminal active site domain typical for class II aaRSs, which comprises the three class II conserved signature motifs, whereas the first 100 N-terminal residues form an antiparallel ␣-helical coiledcoil involved in binding of tRNA [5, 7, 8] and positioning of the 3Ј-end of tRNA in the active site of the C-terminal domain. The x-ray structure of Methanosarcina barkeri SerRS (mMbSerRS) (Fig. 1) [11] revealed that the overall structure of the catalytic module is built from antiparallel strands surrounded by ␣-helices as for the bacterial-type SerRS, the mode of amino acid substrate binding is quite different and involves a zinc ion located in a deep cleft in the active site of the methanogenic-type enzyme [11]. Because the crystal structure of mMbSerRS in the complex with tRNA has not been obtained yet, as it has been for the bacterial system [10], the role of the N-terminal domain in the tRNA binding and aminoacylation is still unclear
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