Team:Bielefeld-CeBiTec/Project/translational system/library and selection

Library and Selection

Short Summary

The incorporation of a non-canonical amino acid (ncAA) requires a tRNA/aminoacyl-tRNA synthetase(tRNA/aaRS) pair which is able to accept and bind the ncAA (to charge the tRNA with the ncAA). The tRNA/aaRS pair should be very specific in the incorporation of the ncAA. Therefore a library of the mutated orthogonal tRNA/aaRS is generated and undergoes numerous rounds of positive and negative selection for the adaption of the codon recognition and the amino acid binding. The selection results in a synthetase which can be expressed efficiently in E.coli and is able to reliable incorporate an unnatural amino acid.

Tyrosy-l and Pyrrolysyl-tRNA Synthetases

Tyrosyl-tRNA Synthetase

There are several amino acids which have been incorporated into peptides and proteins in Escherichia coli . Among others, it was possible to incorporate ncAA with heavy atoms, keto and alkaline side chains, photo crosslinking and so on (Zhang et al.2005). For the binding of the ncAA, an adaption of the synthetase is necessary. This is done by certain mutations of several regions of the synthetase, leading to a conformational change of the binding pocket and resulting in the binding of a new amino acid. The tyrosine synthetase is the first orthogonal E. coli tRNA/aaRS pair generated from archaea and the best known so far (Wang et al.2001). This tyrosine synthetase (TyrRS) has a small anticodon loop binding domain (Steer et al.1999), so the change of the recognition in order to change the anticodon loop of its cognate tRNA to CUA is not as difficult as with a larger anticodon loop binding domain (Steer et al.1999). The binding side is located deep inside a small pocket of the TyrRS (Tian et al.2004). This leads to assume, that no large conformational changes are necessary for the incorporation of the ncAA. Rather very small conformational changes result in a larger or smaller binding pocket and an altered hydrogen-binding interaction with the ligand (Zhang et al.2005). The TyrRS binding pocket is highly hydrophilic, preventing the binding of phenylalanine, whose structure is similar to the one of tyrosine (Goldgur et al.1997). Changing the characteristic of the binding pocket into a hydrophobic kind, could favor the binding of phenylalanine derivates (Goldgur et al.1997). In the middle of the negative charged binding pocket (Zhang et al.2005), there is an aspartic acid on position 158. The entrance of the binding pocket is positioned between two glutamic acids at positions 36 and 172. Upon tRNA binding, the aspartic amino acid Asp158 forms two hydrogen bonds with the glycine at position 34 (Zhang et al.2005).

Figure 1: Tyrosine-binding site in apo M. jannaschii TyrRS
The electrostatic distribution around the tyrosine-binding site is shown. Positive potential is blue (10 mV), neutral potential (0 mV) is white, and negative potential (−10 mV) is red (Zhang et al.2005).

When the ligand tyrosine is bound, hydrogen bonds to the Tyr32, Asp158, Glu36, Gln173, Tyr151 and Gln155 are formed, resulting in subtle movements of the side chains within the tyrosine-binding pocket (Zhang et al.2005). Beside the binding pocket, also other domains are affected by the binding of the tyrosine. For example, the loop 73-83, positioned at the entrance of the binding pocket, which due to the conformational changes then provides a hydrophobic lid over the binding pocket. This is assumed to have the effect of separating the activated tyrosine from water during the catalytic reaction (Zhang et al.2005).

Pyrrolysyl-tRNA Synthetase

In nature one orthogonal aminacyl-tRNA synthetase pair incorporates the 22nd proteinogenic amino acid pyrrolysine (Pyl). Interestingly, Pyl is assigned to the amber stop codon (UAG), thus, it is incorporated naturally through an endogenous nonsense codon suppression (Burke et al., 1998; Paul et al., 2000). It was discovered in the archaeal order of Methanosarcinales (Srinivasan et al., 2002). Due to this specific property the aminoacyl-tRNA synthetase/tRNAPyl pair can be used to expand the genetic code. The pyrrolysyl synthetase (PylRS) is a class II synthetase with a highly conserved class II catalytic core at the C-terminal of around 270 amino acids with a Rossmann fold for ATP-binding. The PylRS forms a obligate dimer with an active site in each subunit (Kavran et al., 2007; Nozawa et al., 2009). Interestingly the N-terminal of the synthetase shows no homology with any known protein domains and its length is variable. The N-terminal leads to aggregation of full-length PylRS due to its high insoluble properties (Yanagisawa et al., 2006; Jiang and Krzycki, 2012). Therefore a truncated version containing the catalytic core of the synthetase is used (residues 185-454) (Kavran et al., 2007; Yanagisawa et al., 2008a). In M. mazei the catalytic core of PylRS has a deep hydrophobic pocket where A302, L305, Y306, L309, N346, C348 and W417 form a large cavity for the binding of the Pyl side chain ((4R,5R)-4-methyl-pyrroline-5-carboxylate) (Kavran et al., 2007; Yanagisawa et al., 2008a). This hydrophobic interactions are relatively non-specific and given that the PylRS does not have an editing domain for hydrolysation of misacylated tRNAs this synthetase shows high substrate side chain promiscuity (Wan et al., 2014). On top of that Pyl is quite large in comparison to the other endogenous canonical amino acids, thus there was no selection pressure to stringently recognize Pyl for the PylRS (Wan et al., 2014). Therefore structurally similar and dissimilar non-canonical amino acids were shown to be incorporated using PylRS (Mukai et al., 2008; Polycarpo et al., 2006; Yanagisawa et al., 2008b). in the active site of PylRS Y384 and N346 form hydrogen bond interactions with the α-amine of Pyl (Yanagisawa et al., 2008a; Kavran et al., 2007). But PylRS also has a high tolenrance for α-hydroxy acids (Kobayashi et al., 2009; Li et al., 2012).

Figure 2: Pyrrolisine-binding site of WT Metanosarcina mazei. Strucutral composition of the active site of PylRS with activated Pyl (Pyl-AMP) as a substrate (Guo et al., 2014).

Most interestingly the PylRS does not show a direct synthetase-anticodon interaction as other synthetases do. Even a mutation at the anticodon of tRNAPyl does not influence aminoacylation activity of the PylRS (Ambrogelly et al., 2007; Yanagisawa et al., 2008a). This characteristic can be used to incorporate non-canonical amino acids using unnatural base pairs. The only drawback using PylRS is its low translational efficiency and catalytic activity compared to other synthetases (Guo et al., 2014). Because in nature PylRS is rarely and selectively expressed, a high catalytic and translational efficiency is not needed (Srinivasan et al., 2002). Every change in the native PylRS does lower the catalytic and translational activity (Guo et al., 2014).

Generating the Library

Being able to pass as many as possible mutated synthetases through the cycle of the two selections for the adaption of the specific ncAA, a large library of different synthetase versions has to be created. The large library of random variants of sequences can be generated by side specific mutagenesis using randomized primers with the NNK scheme to avoid the incorporation of stop codons. The randomized N codes for the bases A,C,G,T and the randomized K codes for the bases G,T. This leads to the use of 32 codons, so every codon except the three stop codons and the one start codon is encoded through the NNK scheme (Yuval et al.2011). By comparison to other randomized schemes, the NNK has a relatively low ratio between the most common and rarest coded amino acid (3/32 vs. 1/32) (Yuval et al.2011). By the randomization of more than one position, numerous more sequence variants occur. If one position is randomized, 130 variants of the sequences are needed to attain a 0.99 probability of discovering the best variant (Yuval et al.2011). At the same time, for the randomization of three positions, there are statistically 102,478 variants needed to discover the best variant. In this context, the probability of full codon coverage changes from 0.82 % for one randomizes position to 3.25 • 10-36 % for three randomized position (Yuval et al.2011). Regarding, that not the full coverage is essential, but rather the discovering of the best variant, the NNK is a solid method for the generation of a synthetase library for the selection of the best ncAA binding site.

Selection

For the translational incorporation of ncAAs, a corresponding tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pair is essential. The tRNA/aaRS pair must recognize and deliver the novel amino acid in response to a unique codon that does not encode any of the 20 canonical amino acids. This leads to a modification of the tRNA for the recognition of the amber-codon and to a modification of the synthetase for the specific binding of a new amino acid. To ensure specificity and orthogonality, a tRNA/aaRS pair from a distinct microbial organism is used to generate tRNA and aaRS libraries which are used to evolve a suitable tRNA/aaRS pair in iterative negative and positive selection steps (Wang et al.2006). Therefore the tRNA and the aaRS each have to be separately mutated and selected.

Modification of the aaRS

In the negative selection step, the synthetase (aaRS) library is tested for orthogonality of the exogenous tRNAs towards the endogenous translation system. The expression of a barnase gene is prevented by an amber stop codon in its coding region. If the exogenous synthetase binds an endogenous amino acid, the amber stop codon preventing the expression of barnase is suppressed, which leads to cells death. Therefore, only cells with orthogonal or non-functional synthetases survive (Wang et al.2006, Liu et al.2010). The orthogonal aaRS are then subjected to positive selection, in which the tRNA/aaRS pairs are tested for their functionality. The expression of an antibiotic resistance gene is prevented by an amber stop codon. Only if the exogenous aaRS incorporates any amino acid, the amber stop codon is suppressed and the cell attains antibiotic resistance. Resulting in the death of cells who do not own a functional aaRS (Wang et al.2006, Liu et al.2010).

Figure 2: Adaption of the aminoacyl-synthetase.
Positive and negative selection for the specificity of the orthogonal synthetase for the ncAA in E. coli.

Modification of the tRNA

Similar to the adaption by selection of the aaRS is the selection of a proper variant of the tRNA. Regarding the unnatural base pairs (UBP), the recognition of the unnatural bases through the tRNA for the incorporation of the non-canonical amino acids requires significant modifications of the tRNA. The recognition of the amber stop codon has already been achieved in other laboratories (Wang et al.2006, Liu et al.2010). Therefore, a selection cycle of negative and positive selections similar to the one of the aaRS, mentioned above, was performed. The main difference is the exchange of the tRNA by the aaRS on the plasmids used for the selection (Wang et al.2006, Liu et al.2010). The outcome is a tRNA, only able to recognize the amber codon and at the same time recognizes its orthogonal synthetase. The combination of the tRNA and aaRS modification results in an orthogonal tRNA/aaRS pair capable of specifically incorporating a certain ncAA if the amber codon is present.

References

Yuval Nov, (2011). When Second Best Is Good Enough: Another Probabilistic Look at Saturation Mutagenesis. Applied and Environmental Microbiology 099-2240/12/12.00.
Zhang Y, Wang L, Schultz PG, Wilson IA. (2005). Crystal structures of apo wild-type M. jannaschiityrosyl-tRNA synthetase (TyrRS) and an engineeredTyrRS specific for O-methyl-L-tyrosine. Protein Sci 14(5): 1340–1349
Wang L, Xie J, Schultz PG. (2006). Expanding the Genetic Code. Annu. Rev. Biophys. Biomol. Struct. 2006;35:225–49.
Liu CC, Schultz PG. (2010). Adding New Chemistries to the Genetic Code. Annu. Rev. Biochem. 2010;79:413–44.