Introduction
Histone Methylation
Histone proteins aid in the packing of DNA for the purpose of compacting the genome in the nucleus of the cell and regulating physical accessibility of genes for transcription. The protein itself is an octamer made of heterodimer core proteins H2a, H2b, H3, and H4, with H1 and H5 acting as linker proteins. About 145-157 base pairs wind around a histone core protein. (1) Modifications to histone core proteins can affect the accessibility of genes in the genome and their ability to be transcribed. Some of these modifications include methylation/demethylation, acetylation/deacetylation, and ubiquitination/deubiquitination. (2)
Specifically, histone methylation is associated with gene activation. (3) Many domain families fall under the Histone methylase family, one of these enzymes being the family, which can target H3, H4, or H2a; each of these methylation sites can have different effects on gene expression within the genome. Typically, methylation of some of these sites are always present on both active and inactive genes, extra methylations required for activity. (4) Some tumor related genes such as p53 are site specifically methylated to promote biological function (5), whereas hypomethylation of CpG is linked to tumor genesis. (2) A particular enzyme in the SET7 domain family is lysine methyltransferase, which acts on the histone by adding a methyl group to Lys4 on H3; the addition results in promotion of gene unwinding and gene transcription. (4,3)
KMT Structure
Composition
The secondary structure is composed of 10% and 37% . The helical composition includes 3 , with two residing in the SET domain and one in the C-terminal domain. The alpha helices in the SET domain are two turns while the C-terminal helix is by far the largest with 4 turns. There are also 2 in the SET domain which are each one turn. There are 21 total beta strands which reside in the N-terminal domain and the SET domain. The beta strands are primarily anti-parallel and multiple antiparallel strands are connected by and beta turns.
The N-terminal Domain
The N-terminal domain has no notable function in regard to the activity of KMT; however, it has been related to stability of the enzyme and most likely prevents its degradation within the cell. The N-terminal domain is highly conserved across species, which supports of the claim of necessity for stability. A Clustal alignment of a portion of the region shows a variety of high level species with conservation.
The C-Terminal Domain
The of lysine methyltransferase is essential for the catalytic activity of the enzyme. Hydrophobic packing of the C-terminal segment (residues 345-366) forms the lysine access channel. Residues 337-349 create a that stabilizes the orientation of two tyrosine residues Tyr 335 and Tyr337 that form the lysine access channel. The hydrophobic packing of the C-terminal against beta sheet 19 (specifically residue 299) orient the SAM cofactor so the methyl donating group is oriented toward the lysine access channel. donating group is oriented toward the lysine access channel.
The Active Site
The active site and binding pocket of KMT have several essential characteristics for the overall efficiency. First, the lysine of the histone enters the active site “with difficulty” which is facilitated by the faces of flanking . Once in the active site, the alkyl part of the histone chain is stabilized by the , and polar residues are stabilized by hydrogen bonding interactions on the surface. The Y335 and Y337 are also essential for stabilization of histone chain via hydrogen bonding.
The itself contains the cofactor S-adenosyl methionine (SAM) which donates the methyl group in the reaction. (4)
S-adenosyl homocysteine (SAH)
The reaction is catalyzed by Y305, Y245, carbonyl oxygens of the main chain in residues 295 and 290. Y305 and the carbonyl oxygens stabilize and pull electron density off a water to pull on one of the hydrogens off the nitrogen of the lysine, while oxygen of Y245 pulls on the other hydrogen of the nitrogen. Both of these actions allow nitrogen to become more nucleophilic and attack the carbon of the methyl group on the SAM, which is attached to a positively charged sulfur. The methyl group is then transferred and the sulfur is neutral; SAM has been converted to S-adenosyl homocysteine (SAH). (4)
Inhibitors
Sinefungin is a potent methyltransferase inhibitor. It is a structural analog of S-adenosylmethionine that is more stable due to the ability to create two additional hydrogen bonds to its amine group in the active site. It has been used experimentally to inhibit the SET 7/9 protein on peritoneal fibrosis in mice and in human peritoneal mesothelial cells. Tamura et al. (2018) found that sinefungin suppressed the cell accumulation and thickening in methylglyoxal peritoneal fibrosis.
References
1. DesJarlais, R.; Tummino, P.J. Role of Histone-Modifying Enzymes and Their Complexes in Regulation of Chromatin Biology. Biochemistry 2016, 55: 1584-1599.
2. Lu, D. Epigenetic modification enzymes: catalytic mechanisms and inhibitors. Acta Pharmaceutica Sinica B 2013, 3: 141-149.
3. Dong, X.; Weng, Z. The correlation between histone modifications and gene expression. Epigenomics 2013, 5: 113-6.
4. Xiao, B.; Jing, C.; Wilson, J.; Walker, P.; Vasisht, N.; et al. Structure and catalytic mechanism of the human histone methyltranferase SET7/9. Letters to Nature 2003, 412: 652-655.
5. Del Rizzo, P. A.; Trievel, R.C. Substrate and product specificities of SET domain methyltransferases. Epigenetics 2011, 6: 1059-1067
6. Mao, X.; Qiao, Z.; Fan, C.; Guo, A.; Yu, X.; Jin, F. Expression pattern and methylation of estrogen receptor α in breast intraductal proliferative lesions. Oncology Reports 2016, 36: 1868-1874.
7. Tamura, R., Doi, S., Nakashima, A., Sasaki, K., Maeda, K., Ueno, T., & Masaki, T. (2018). Inhibition of the H3K4 methyltransferase SET7/9 ameliorates peritoneal fibrosis. PloS one, 13(5), e0196844. doi:10.1371/journal.pone.0196844
8. Schluckebier et al. (1997), Differential binding of S-andeosylmethionine S-adenosylhomocysteine and Sinefungin to adenine-specific DNA methyltransferase M. TaqI; J. Mol. Biol., 265 56
