Synthesis of ε-N-propionyl-, ε-N-butyryl-, and ε-N-crotonyl-lysine containing histone H3 using the pyrrolysine system

Abstract

Recently new lysine modifications were detected in histones and other proteins. Using the pyrrolysine amber suppression system we genetically inserted three of the new amino acids e-N-propionyl-, e-N-butyryl-, and e-N-crotonyl-lysine site specifically into histone H3. The lysine at position 9 (H3 K9), which is known to be highly modified in chromatin, was replaced by these unnatural amino acids.The histone code is based on the post-translational modification of critical amino acid residues in different histones. Among these, lysine acetylations and methylations are the most abundant and affect the transcriptional status of the genes associated with the corresponding histones. [1][2][3] The role of these modifications is sequence dependent. Typically acetylation is associated with transcriptionally active genes, while methylation induces transcriptional silencing. 4,5 Recently Zhao and co-workers discovered a number of new modified amino acids in histones. [6][7][8] Some of these were also detected in proteins other than histones, raising the possibility that they are of more widespread importance. [9][10][11] The new post-translational modifications (PTMs) are acylated derivatives of lysine at the e-amino position. The acylation partners are propionic acid, butyric acid, malonic acid, succinic acid, crotonic acid or fatty acids. A common characteristic of these compounds is that they are key metabolic intermediates that typically exist as CoA activated thioester species in the cell. 12 It is currently not clear how these newly discovered PTMs are biosynthetically established within the histones and we do not fully understand if and how they influence genetic processes.So far no specific deacylases for e-N-propionyl-or e-Nbutyryl-lysine have been identified. However Sirt5, a member of NAD-dependent sirtuins is able to specifically deacylate e-Nmalonyl-and e-N-succinyllysine. 11 Recently it was discovered that HDAC3 exhibits decrotonylase activity in vitro 13 and it was found that lysine crotonylation activates genes even in a globally repressive environment. 7,14 In order to enable investigation of the new lysine derivatives in histones it is essential to generate histones that contain these amino acids site specifically. 15 In this direction semi-synthetic chemical ligation based methods 16,17 were utilized and chemical methods were employed to generate acetyllysine (Kac) and methylated-lysines or derivatives thereof. [18][19][20][21] Chin and co-workers reported the introduction of Kac into H3 K56 22 using the pyrrolysine system. This system was also employed for the synthesis of monomethyl-and dimethyl-lysine containing histones. 23,24 Schultz et al. recently described the synthesis of histone H2B containing e-N-crotonyllysine at position K11 using an evolved pyrrolysyl-tRNA synthetase from Methanosarcina barkeri. 25 Here we show that the pyrrolysine system can be used to insert e-N-propionyl-(Kpr, 1), e-N-butyryl-(Kbu, 2), and e-Ncrotonyl-lysine (Kcr, 3) into histones at critical positions such as H3 K9 using...

Molecular Coupling of Histone Crotonylation and Active Transcription by AF9 YEATS Domain

“…Recombinant pre-modified H3K9ac, H3K9cr, H3K18ac, and H3K18cr histones were generated by amber suppression, as previously described (Gattner et al, 2013; Kim et al, 2012) and validated by MS/MS analysis to be >90% modified (Figures 3C and S3B). These pre-modified H3 histones, in conjunction with unmodified recombinant core histones, were assembled into nucleosomes by gradient salt dialysis (Dyer et al, 2004) with a biotinylated 601 DNA template, allowing the nucleosomes to be immobilized by streptavidin (Figures S3C and S3D).…”

The use of unnatural amino acids to study and engineer protein function

“…The biological consequences of these modifications are largely unknown. Amber suppression provides the tools for introducing a subset of these modifications in recombinant proteins, which will facilitate their biochemical characterization [25][26][27][28][29].…”

Pyrrolysyl-tRNA synthetase: An ordinary enzyme but an outstanding genetic code expansion tool

“…[81, 82] Recently Carell et al showed that PylRS weakly recognized three NCAAs ( 20 – 22 ) and mediated their incorporation into proteins at an amber codon in coordination with tRNA Pyl , allowing the synthesis of proteins with site-specific lysine propionylation, butylation, and crotonylation. [83] PylRS mutants with much improved binding affinities toward 20 – 22 were developed concurrently in the Schultz group and in our group. They will be discussed in the next section.…”