Incorporation of Uaas into proteins using a host’s endogenous translation machinery opens the door to addressing questions with chemical precision that is unattainable using naturally occurring amino acids. This expanded toolset allows one to pose and answer more in-depth molecular questions without the limitations imposed by the 20 natural amino acids used in traditional mutagenic analyses.1,2
Aminoacyl-tRNA synthetases (RSs) obtained by structure-based engineering and directed evolution efficiently recognize and activate Uaas through ATP-dependent adenylation and subsequently catalyze transfer to their cognate tRNA. To date, more than 70 Uaas are now amenable to translational insertion into proteins in bacteria, yeast, or mammalian cells using these artificially evolved tRNA/RS pairs.(3
) By choosing particular matching sets of tRNA/RSs from diverse organisms, the pairs can function in vivo
in an orthogonal manner. In other words, there is limited if any crosstalk between the expression host’s native tRNA/RS pairs and the orthogonal tRNA/RS pair; however, the orthogonal pair is still able to functionally couple with the host’s protein translational machinery. The tRNATyr
/TyrRS pair derived from the archaeon Methanocaldococcus jannaschii
was the first successful pair to direct the genetically encoded incorporation of the Uaa O
-tyrosine (Ome) into translated proteins in vivo
This initial result then spawned the incorporation of the majority of Tyr-based Uaas possessing distinct functional groups into recombinant proteins. Recently, another matching set, namely, the tRNAPyl
/PylRS pair, was used to incorporate Uaas bearing functional groups similar to Pyl.6−12
In most cases regarding tRNATyr
/TyrRS systems, sterically innocuous modifications of the phenyl ring, typically focused on the para
position, are employed as unnatural substrates. Similarly, most Uaas incorporated by the tRNAPyl
/PylRS pair all contain slight variations of the extended Pyl side chain, while the core Lys moiety and the Nε
-carbonyl group of Pyl remain unchanged.(3
) In short, most evolved orthogonal tRNA/RS pairs demonstrate specificity for Uaas that maintain the chemical core of the native amino acid substrate, thus limiting the stereochemical diversity of Uaas accessible to the researcher. Since the RS component of the pair plays a key role in amino acid substrate selection, it is of tremendous importance to probe the extent to which it can be engineered not only to activate a chemically and structurally divergent amino acid but also its ability to transfer this new amino acid to its cognate tRNA. Given that the degree of substrate specificity exhibited by an RS is functionally coupled to the accuracy of protein translation, the RS is restrained by tremendous selective pressure; therefore, integration of structural approaches with directed evolution of RSs can expand upon our understanding of the evolutionary tenets governing substrate specificity and selection.13−15
This unified approach applied in the form of multiple rounds of structure-based design and directed evolution can often produce RSs with altered specificity for Uaa substrates in relatively rapid fashion.
Moreover, the inability to efficiently employ mammalian cells and multicellular organisms for evolving tRNA/RS pairs further limits the scope of unnatural substrates available for probing in vivo
protein function in organisms more complex than microbial hosts. Currently, directed approaches for evolving a Uaa-specific RS involve the generation of an initial RS mutant library minimally containing 108
These numbers, coupled with the need for efficient selection procedures, limit applicability to organisms possessing high transformation efficiency and favorable growth characteristics.(1
) The approach was first developed in E. coli
) and later expanded to include another unicellular organism, Saccharomyces cerevisiae
Nevertheless, maintenance of large mutant libraries and selection methods are difficult to implement in more complex systems such as mammalian cells and multicellular organisms.
One solution is to evolve an RS that possess characteristics favorable to deployment in the intended mammalian hosts in recombinant organisms such as E. coli
or yeast. Assuming the appropriate RS is chosen, the evolved RSs should then be readily integrated into mammalian cells or a multicellular organism for further optimization once the large collection of mutants is narrowed substantially. This divide and conquer approach thus shortcuts some of the anticipated problems faced during the initial rounds of selection.(19
) This kind of strategy was applied to the tRNATyr
/TyrRS pair from E. coli
, wherein the TyrRS was first evolved in yeast and then transferred to mammalian cell hosts.19,20
Nonetheless, most reported mutant RSs evolved in yeast generally lack the in vivo
efficiency of the original M. jannaschii
TyrRS evolved and exploited in E. coli
. Moreover, M. jannaschii
TyrRS does not operate in an orthogonal manner in mammalian cells, often cross-charging mammalian tRNAs.
With these limitations in mind, tRNAPyl
/PylRS is an attractive alternative because of the demonstrated orthogonal behavior of this particular pair in E. coli
and mammalian cells.9,10,21,22
Mutating the active site does not impact orthogonality so the evolved pairs are highly unlikely to cross-react with the endogenous tRNA/RS pairs of their mammalian hosts. One remaining issue, however, concerns the extent to which the current assortment of orthogonal pairs can be evolved to specifically adenylate and then charge their cognate tRNAs with substrates substantially different from their natural substrates. Strikingly, to date, Uaas incorporated by mutant RSs derived from PylRS bear close chemical and steric resemblance to the natural Pyl substrate.(3
High fidelity Uaa incorporation is essential for generating chemically homogeneous proteins for investigation. An optimally evolved RS should function similarly to a wild-type RS, which achieves high incorporation fidelity under physiological growth conditions and maintains fidelity when cells are grown in nutrient-rich media. For Uaa incorporation in E. coli
, nutrient-rich media is necessary to ensure high in vivo
incorporation efficiency of the evolved RS; however, when an evolved RS possesses non-optimal specificity for the Uaa, minimal media lacking certain or all natural amino acids must be used to limit the mis-incorporation of any natural amino acids into the recombinant protein.(23
) Unfortunately, minimal media cannot be applied to traditional fermentations, mammalian cell cultures, and whole organism engineering; this drawback prevents the transfer and usage of non-optimally evolved RSs in these situations. Moreover, the active site of a non-optimal RS must lack the complementary stereochemical features for selection of the given Uaa substrate. Structural analyses then become critical for providing architectural guides to explain and further optimize the substrate selection and turnover. Recently, Wang et al.
reported the incorporation of two Phe-based Uaas into proteins via
evolved PylRSs, albeit in E. coli
grown solely in minimal media.(24
) To date, efficient evolution of PylRS to specifically incorporate a Uaa significantly deviating from the native Pyl in rich media followed by transfer to mammalian hosts has not been demonstrated. Moreover, the structural transformations that occur in the active site of such a highly specific PylRS mutant necessary for accommodating a dramatic substrate change remain unclear.
Here we show that the Methanosarcina mazei PylRS (MmPylRS) can be evolved to efficiently charge a Uaa with a short aromatic side chain, in contrast to the long aliphatic side chain of the native substrate Pyl. The evolved RS incorporates the Uaa into proteins with high fidelity in both E. coli grown in rich media and mammalian cells. Additionally, we solved and refined the X-ray crystal structure of the evolved PylRS complexed with the new Uaa and an ATP analogue to a nominal resolution of 1.75 Å. The three-dimensional structure and active site architecture in the trapped substrate-bound form confirms that the mutations obtained contribute directly to the high stereochemical selectivity of our mutant PylRS for turnover of Tyr-based Uaas. Furthermore, this combined structure–function investigation provides the experimental support necessary to continue diversifying and optimizing PylRS variants in E. coli to further expand the variety of smaller Uaas for later use in mammalian cells.