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.
Highlights
The regulation of many cellular key processes, such as gene expression, protein activity and stability as well as molecular recognition relies on the posttranslational modification (PTM) of proteins
Despite the fact that some of the lysine modifications like buturylation, crotonylation and propionylation are very similar in their chemical structure (Fig. 1), their effect on gene expression differ depending on the context
Lysine PTMs are not restricted to histones – for example, the tumor suppressor protein p53 is regulated by phosphorylation and ubiquitination at its C-terminal lysine residues [14,15]
Summary
The regulation of many cellular key processes, such as gene expression, protein activity and stability as well as molecular recognition relies on the posttranslational modification (PTM) of proteins. It became clear that lysine residues in histones can be acetylated or methylated, and malonylated, succinylated, butyrylated, propionylated and crotonylated [4,5,6,7,8,9] These modifications change the net charge of the residue from positive to negative or neutral as well as alter the hydrophobicity and flexibility of the modified protein. 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- allowing the functionalization of the target protein [16,17,18,19,20,21,22,23,24]. The wild type PylRS from M. mazei, which shares doi:10.1371/journal.pone.0096198.g002
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