The genetic encoding of synthetic or "non-natural" amino acids promises to diversify the functions and structures of proteins. We applied rapid codon-reassignment for creating Escherichia coli strains unable to terminate translation at the UAG "stop" triplet, but efficiently decoding it as various tyrosine and lysine derivatives. This complete change in the UAG meaning enabled protein synthesis with these non-natural molecules at multiple defined sites, in addition to the 20 canonical amino acids. UAG was also redefined in the E. coli BL21 strain, suitable for the large-scale production of recombinant proteins, and its cell extract served the cell-free synthesis of an epigenetic protein, histone H4, fully acetylated at four specific lysine sites.
Post-translational modifications (PTMs) of histones, such as lysine acetylation of the N-terminal tails, play crucial roles in controlling gene expression. Due to the difficulty in reconstituting site-specifically acetylated nucleosomes with crystallization quality, structural analyses of histone acetylation are currently performed using synthesized tail peptides. Through engineering of the genetic code, translation termination, and cell-free protein synthesis, we reconstituted human H4-mono- to tetra-acetylated nucleosome core particles (NCPs), and solved the crystal structures of the H4-K5/K8/K12/K16-tetra-acetylated NCP and unmodified NCP at 2.4 Å and 2.2 Å resolutions, respectively. The structure of the H4-tetra-acetylated NCP resembled that of the unmodified NCP, and the DNA wrapped the histone octamer as precisely as in the unmodified NCP. However, the B-factors were significantly increased for the peripheral DNAs near the N-terminal tail of the intra- or inter-nucleosomal H4. In contrast, the B-factors were negligibly affected by the H4 tetra-acetylation in histone core residues, including those composing the acidic patch, and at H4-R23, which interacts with the acidic patch of the neighboring NCP. The present study revealed that the H4 tetra-acetylation impairs NCP self-association by changing the interactions of the H4 tail with DNA, and is the first demonstration of crystallization quality NCPs reconstituted with genuine PTMs.
The BET family proteins recognize acetylated chromatin through their two bromodomains, acting as transcriptional activators or tethering viral genomes to the mitotic chromosomes of their host. The structural mechanism for how the N-terminal bromodomain of human BRD2 (BRD2-BD1) deciphers the mono-acetylated status of histone H4 tail was recently reported. Here we show the crystal structure of the second bromodomain of BRD2 (BRD2-BD2) in complex with the di-acetylated histone H4 tail (H4K5ac/K12ac). To our surprise, a single K5ac/K12ac peptide interacts with two BRD2-BD2 molecules simultaneously: the K5ac residue binds to one BRD2-BD2 molecule while the K12ac residue binds to another. These results provide a structural basis for the recognition of two different patterns of the histone acetylation status by a single bromodomain.
Structured summary
MINT-7989882, MINT-7989824, MINT-7989846, MINT-7989865: H4 (uniprotkb:P62805) binds (MI:0407) to BRD2 (uniprotkb:P25440) by surface plasmon resonance (MI:0107)
MINT-7989539: H4 (uniprotkb:P62805) and BRD2 (uniprotkb:P25440) bind (MI:0407) by X-ray crystallography (MI:0114)
Lysine methylation is one of the important post-translational modifications of histones, and produces an N(ε) -mono-, di-, or trimethyllysine residues. Multiple and site-specific lysine methylations of histones are essential to define epigenetic statuses and control heterochromatin formation, DNA repair, and transcription regulation. A method was previously developed to build an analogue of N(ε)-monomethyllysine, with cysteine substituting for lysine. Here, we have developed a new method of preparing histones bearing multiple N(ε)-monomethyllysine residues at specified positions. Release factor 1-knockout (RFzero) Escherichia coli cells or a cell-free system based on the RFzero cell lysate was used for protein synthesis, as in RFzero cells UAG is redefined as a sense codon for non-canonical amino acids. During protein synthesis, a tert-butyloxycarbonyl-protected N(ε)-monomethyllysine analogue is ligated to Methanosarcina mazei pyrrolysine tRNA (tRNA(Pyl)) by M. mazei pyrrolysyl-tRNA synthetase mutants, and is translationally incorporated into one or more positions specified by the UAG codon. Protecting groups on the protein are then removed with trifluoroacetic acid to generate N(ε)-monomethyllysine residues. We installed N(ε)-monomethyllysine residues at positions 4, 9, 27, 36, and/or 79 of human histone H3. Each of the N(ε)-monomethyllysine residues within the produced histone H3 was recognized by its specific antibody. Furthermore, the antibody recognized the authentic N(ε)-monomethyllysine residue at position 27 better than the N(ε)-monomethyllysine analogue built with cysteine. Mass spectrometry analyses also confirmed the lysine modifications on the produced histone H3. Thus, our method enables the installation of authentic N(ε)-monomethyllysines at multiple positions within a protein for large-scale production.
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