Stereochemical Basis for Engineered Pyrrolysyl-tRNA Synthetase and the Efficient in Vivo Incorporation of Structurally Divergent Non-native Amino Acids

The Jack H. Skirball Center for Chemical Biology & Proteomics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, United States.
ACS Chemical Biology (Impact Factor: 5.33). 05/2011; 6(7):733-43. DOI: 10.1021/cb200057a
Source: PubMed


Unnatural amino acids (Uaas) can be translationally incorporated into proteins in vivo using evolved tRNA/aminoacyl-tRNA synthetase (RS) pairs, affording chemistries inaccessible when restricted to the 20 natural amino acids. To date, most evolved RSs aminoacylate Uaas chemically similar to the native substrate of the wild-type RS; these conservative changes limit the scope of Uaa applications. Here, we adapt Methanosarcina mazei PylRS to charge a noticeably disparate Uaa, O-methyl-l-tyrosine (Ome). In addition, the 1.75 Å X-ray crystal structure of the evolved PylRS complexed with Ome and a non-hydrolyzable ATP analogue reveals the stereochemical determinants for substrate selection. Catalytically synergistic active site mutations remodel the substrate-binding cavity, providing a shortened but wider active site. In particular, mutation of Asn346, a residue critical for specific selection and turnover of the Pyl chemical core, accommodates different side chains while the central role of Asn346 in aminoacylation is rescued through compensatory hydrogen bonding provided by A302T. This multifaceted analysis provides a new starting point for engineering PylRS to aminoacylate a significantly more diverse selection of Uaas than previously anticipated.

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Available from: Nikki Dellas, Sep 18, 2014
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    • "By introducing an amber stop codon in a given gene, the host's endogenous translational machinery can be employed to incorporate non-natural amino acids site-specifically, thus allowing the functionalization of the target protein [16], [17], [18], [19], [20], [21], [22], [23], . Structures of PylRS in complex with its natural substrate [25], [26], [27] as well as evolved mutants containing non-natural amino acids are already available [28], [29], [30]. Using an evolved PylRS (L274A C313A Y349F) from M. barkeri ε-N-crotonlyl-lysine was introduced into histones [17]. "
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    ABSTRACT: Posttranslational modifications (PTMs) of proteins determine their structure-function relationships, interaction partners, as well as their fate in the cell and are crucial for many cellular key processes. For instance chromatin structure and hence gene expression is epigenetically regulated by acetylation or methylation of lysine residues in histones, a phenomenon known as the 'histone code'. Recently it was shown that these lysine residues can furthermore be malonylated, succinylated, butyrylated, propionylated and crotonylated, resulting in significant alteration of gene expression patterns. However the functional implications of these PTMs, which only differ marginally in their chemical structure, is not yet understood. Therefore generation of proteins containing these modified amino acids site specifically is an important tool. In the last decade methods for the translational incorporation of non-natural amino acids using orthogonal aminoacyl-tRNA synthetase (aaRS):tRNAaaCUA pairs were developed. A number of studies show that aaRS can be evolved to use non-natural amino acids and expand the genetic code. Nevertheless the wild type pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of non-natural amino acids. Here we present structural data on the wild type enzyme in complex with adenylated ε-N-alkynyl-, ε-N-butyryl-, ε-N-crotonyl- and ε-N-propionyl-lysine providing insights into the plasticity of the PylRS active site. This shows that given certain key features in the non-natural amino acid to be incorporated, directed evolution of this enzyme is not necessary for substrate tolerance.
    Full-text · Article · Apr 2014 · PLoS ONE
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    • "However, the Pyl-tRNA is not hardwired for amber codon suppression, and can be adapted to decode a number of other codons (Ambrogelly et al., 2007). In addition, the PylRS has been shown to readily accept a variety of side chain structures (Polycarpo et al., 2006;Yanagisawa et al., 2008;Neumann et al., 2008;Chen et al., 2009;Nguyen et al., 2009;Li et al., 2009;Hancock et al., 2010;Ou et al., 2011;Plass et al., 2011;Takimoto et al., 2011) as well as a set of non-alpha amino derivatives (Kobayashi et al., 2009). This feature makes the PylRS/tRNA pair an ideal candidate for site specific integration of NNAAs. "

    Full-text · Chapter · Feb 2012
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    ABSTRACT: Posttranslational modifications modulate the activities of most eukaryotic proteins and play a critical role in all aspects of cellular life. Understanding functional roles of these modifications requires homogeneously modified proteins that are usually difficult to purify from their natural sources. An emerging powerful tool for synthesis of proteins with defined posttranslational modifications is to genetically encode modified amino acids in living cells and incorporate them directly into proteins during the protein translation process. Using this approach, homogenous proteins with tyrosine sulfation, tyrosine phosphorylation mimics, tyrosine nitration, lysine acetylation, lysine methylation, and ubiquitination have been synthesized in large quantities. In this review, we provide a brief introduction to protein posttranslational modifications and the genetic noncanonical amino acid (NAA) incorporation technique, then discuss successful applications of the genetic NAA incorporation approach to produce proteins with defined modifications, and end with challenges and ongoing methodology developments for synthesis of proteins with other modifications.
    Full-text · Article · Nov 2010 · Molecular BioSystems
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