Structural Characterization of Rosetta Designed Amino Acyl‐tRNA Synthetase Active Sites for Genetic Code Expansion

Abstract

The site‐specific incorporation of non‐standard and novel amino acid structures enabled by Genetic Code Expansion (GCE) provides access to new avenues of protein research allowed by engineering diverse functionality into proteins. At its core, GCE requires the engineering of an orthogonal amino acyl‐tRNA synthetase/tRNA cognate pair that can function with high catalytic efficiency for a non‐canonical amino acid (ncAA). While the GCE community has generated many new amino acyl‐tRNA synthetase/tRNA pairs, these pairs have kinetics that are orders of magnitude worse than the natural translational components. A major challenge for the GCE field is identifying which amino acyl‐tRNA synthetase residues to target for engineering improved amino acyl‐tRNA synthetase/tRNA pairs.Here we use Rosetta modeling to identify mutations in the amino acyl‐tRNA synthetase active site that will improve the efficiency of the nitroTyr amino acyl‐tRNA synthetase/tRNA pairs. Rosetta guided mutations were successful in generating improved GCE function in vivo.Crystal structures were obtained for the Rosetta improved amino acyl‐tRNA synthetases to understand the structural basis for the enhanced activity as part of a comprehensive kinetic and structural analysis of these improved synthetases. Here, we used the 3‐nitro‐tyrosine amino acyl‐tRNA synthetase/tRNA pair as a model system for synthetase improvement and structural characterization.We observe that the Rosetta‐improved synthetases allow a more planar 3‐nitro‐tyrosine (nitroTyr) amino acid upon binding, which is predicted to contribute to their increased activity. This Rosetta guided approach provides a third generation of more efficient nitroTyr amino acyl‐tRNA synthetase/tRNA pairs that will accelerate the study of this oxidative stress post‐translational modification and its role in human disease.We demonstrate that Rosetta successfully identified key residues for amino acyl‐tRNA synthetase/tRNA pair optimization. This strategy can be utilized to systematically improve the efficiency of all ncAA amino acyl‐tRNA synthetase/tRNA pairs, resulting in more robust GCE technologies and facilitating new applications.Support or Funding InformationNIH Grant #R01‐GM114653‐01, Oregon State University Summer Undergraduate Research Experience (SURE) in Science ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.