C-terminal evolutionary remodelling of isoleucyl-tRNA synthetases is a prokaryote-specific strategy for tuning aminoacylation rate

Abstract Aminoacyl-tRNA synthetases are the guardians of translational fidelity. Their complex function is mirrored by an elaborate structure, which includes multiple nested domains. While the evolutionary pressures that promoted the emergence of some domains, such as the editing domain, are clear, the pressures acting on other domains, particularly those at the C-terminus, are not. Here, we use a combination of kinetic analysis, X-ray crystallography, and bioinformatics to unveil the history and evolutionary forces that have shaped isoleucyl-tRNA synthetase (IleRS) domain structure. We find that the traditional classification into IleRS1 and IleRS2, based on the C-terminal tRNA-recognition domains, is incomplete, as it fails to capture features of the synthetic domain. Guided by the crystal structure of the Priestia megaterium IleRS2:tRNA complex, we removed key interactions between IleRS2 and its cognate tRNA and characterised their impact on enzyme activity. We found that D-loop interactions with the IleRS2 C-terminal region are non-essential in prokaryotes, and their loss can even increase catalytic turnover. Further, the zinc-binding domain of IleRS1 recognises the anticodon less stringently than the canonical C-terminal domain of IleRS2. Our data suggest that C-terminal evolutionary remodelling of IleRSs is an ongoing process with a historical precedent, consistent with selection for faster aminoacylation rate.