Structural robustness affects the engineerability of aminoacyl-tRNA synthetases for genetic code expansion

Abstract

The ability to engineer the substrate specificity of natural aminoacyl-tRNA synthetase/tRNA pairs facilitates the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. The Methanocaldococcus jannaschii derived tyrosyl-tRNA synthetase (MjTyrRS)/tRNA pair has been engineered to incorporate numerous ncAAs into protein expressed in bacteria. However, it cannot be used in eukaryotic cells due to cross-reactivity with its host counterparts. The E. coli derived tyrosyl-tRNA synthetase (EcTyrRS)/tRNA pair offers a suitable alternative to this end, but a much smaller subset of ncAAs has been genetically encoded using this pair. Here we report that this discrepancy, at least partly, stems from the lower structural robustness of EcTyrRS relative to MjTyrRS. We show that engineered TyrRS mutants in general exhibit significantly lower thermostability relative to their wild-type counterparts. Derived from a thermophilic archaeon, MjTyrRS is a remarkably sturdy protein and tolerates extensive active site engineering without a catastrophic loss of stability at physiological temperature. In contrast, EcTyrRS exhibits significantly lower thermostability, rendering some of its engineered mutants insufficiently stable at physiological temperature. Our observations identify the structural robustness of an aaRS as an important factor that significantly influences how extensively it can be engineered. To overcome this limitation, we have further developed chimeras between EcTyrRS and its homolog from a thermophilic bacteria, which offer an optimal balance between thermostability and activity. We show that the chimeric bacterial TyrRSs show enhanced tolerance for destabilizing active site mutations, providing a potentially more engineerable platform for genetic code expansion.

Broadening the Toolkit for Quantitatively Evaluating Noncanonical Amino Acid Incorporation in Yeast

“…Moreover, while general performance trends are expected to hold upon transferring ncAA incorporation systems between organisms, quantitative differences between organisms can occur, necessitating rigorous characterizations if further SPS implementation is performed in E. coli, mammalian cells, or cells from other organisms. 59,60 Despite these important considerations, our results provide several initial demonstrations that, in yeast, SPSs are a comparable format to DPSs. The indistinguishable ncAA incorporation measurements obtained from SPSs and DPSs suggest that SPSs could be advantageous for applications where the expression of both the OTS and POI are required, but selectable markers are limited.…”

Thermophilic Pyrrolysyl-tRNA Synthetase Mutants for Enhanced Mammalian Genetic Code Expansion

“…This phenomenon is in line with our expectations because the decreased T m values of MbPylRS mutants (Figure S8) probably make them insufficient to maintain stable and thus be vulnerable to degradation at the physiological temperature. Collectively, these results emphasize the potential benefits of using a somewhat thermostable synthetase for enzyme engineering, 38,39 where sufficient structural robustness results in enhanced tolerance for potentially destabilizing but function-imparting mutations for altered substrate specificity.…”

“…28,29 In addition, a chimera combining E. coli tyrosyl-tRNA synthetase and a thermophilic tyrosyl-tRNA synthetase exhibits higher activity at 37 °C than the wild-type progenitors, in addition to showing appropriate thermostability. 39 Inspired by these results, we were interested in finding out whether we could generate a more efficient chimeric PylRS mutant for enhanced eukaryotic GCE. Using the above-described thermostable PylRSs, we first generated two chimeric PylRSs by separately fusing the MtPylRS and Mf PylRS NTDs to the MbPylRS CTD (Figure 5a); the resulting chimeras are designated as tN-bC PylRS and fN-bC PylRS, respectively.…”

A facile platform to engineer E. coli tyrosyl-tRNA synthetase adds new chemistries to the eukaryotic genetic code, including a phosphotyrosine mimic

“…We recently reported that mutants of EcTyrRS often exhibit low thermostability. 32 In particular, pBPARS-1 was found to be largely insoluble under ambient conditions, which likely explains its poor activity. Interestingly, we found that a large fraction of pBPARS-3.1 is found in the soluble fraction of E. coli cell-free extract, while nearly all of pBPARS-1 is found in the insoluble fraction (Figure S4C).…”