Structural Basis for the Site-Specific Incorporation of Lysine Derivatives into Proteins

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 epsilon-N-alkynyl-,epsilon-N-butyryl-,epsilon-N-crotonyl- and epsilon-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

Structural basis for the site-specific incorporation of lysine derivatives into proteins.

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

Comparison of the binding pockets of PylRS and LysRS.

<p>Two-dimensional plot <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096198#pone.0096198-Wallace1" target="_blank">[52]</a> of residues interacting with Lys (A), Pyl (B) and Kalk (C). Van-der-Waals contacts are shown as red half-spheres. Surface representation of the binding pockets of LysRS (D) and PylRS (E) with Lys, Pyl and Kalk, respectively. (F) Superposition of Pyl (grey), Kalk (orange), Kbu (green), Kcr (blue) and Kpr (red). The surface of the binding pocket is shown as mesh, with the charge distribution indicated by coloring (red  =  negative, blue  =  positive). The surrounding residues are drawn as sticks. (PDB codes PylRS: 2Q7H and LysRS: 3A74).</p

Lysine, pyrrolysine and analogs used in this study.

<p>Lysine, pyrrolysine and analogs used in this study.</p

Simulated annealing-omit Fo-<i>D</i>Fc electron density contoured at 2 σ of PylRS in complex with (A) Kalk (orange), (B) Kbu (green), (C) Kcr (blue) and (D) Kpr (red).

<p>The protein is shown as cartoon, overlaid with its semi-transparent surface representation. Amino acids providing key interactions are drawn as sticks, hydrogen-bonds as dashed lines.</p

Cartoon representation of the overall structure of the catalytic domain of PylRS.

<p>(A) Type-II tRNA-synthetase folding topology of the tRNA synthetase domain from PylRS, shown as cartoon representation, overlaid with its semi-transparent surface. The adenylated Kbu is highlighted as green stick model in the active site. (B-F) Zoom in the active site: PylRS in complex with Kalk (orange), Kbu (green), Kcr (blue) and Kpr (red), drawn as stick models. (r. m. s. d ∼0.35 Å). All four non-natural amino acids bind in the same position.</p

Data collection, processing and structure refinement statistics.

<p>Numbers in parentheses correspond to the high resolution shell.</p