Investigatingd-lysine stereochemistry for epigenetic methylation, demethylation and recognition

Abstract

Histone lysine methylation is regulated by N-methyltransferases, demethylases, and N-methyl lysine binding proteins. Thermodynamic, catalytic and computational studies were carried out to investigate the interaction of three epigenetic protein classes with synthetic histone substrates containing l- and d-lysine residues. The results reveal that out of the three classes, N-methyl lysine binding proteins are superior in accepting lysines with the d-configuration.

Lysine Ethylation by Histone Lysine Methyltransferases

“…We then performed comparative enzymatic assays for KMT‐catalyzed methylation (with AdoMet) and ethylation (with AdoEth and AdoSeEth) of synthetic histone peptides by using MALDI‐TOF MS, as recently described; histone H3 1–15 was used for studies with SETD7 (also known as KMT7), G9a (also known as KMT1C and EHMT2), and GLP (also known as KMT1D and EHMT1), and histone H4 13–27 was used for studies with SETD8 (also known as KMT5A). MALDI‐TOF MS data confirmed that human KMTs catalyzed nearly quantitative methylation of histone peptides in the presence of AdoMet: H3K4me, H4K20me, H3K9me3, and H3K9me3 were formed in the presence of SETD7, SETD8, G9a, and GLP, respectively (Figure , top).…”

“…Expression and purification of KMTs : The four wild‐type human KMTs (SETD7, SETD8, G9a, and GLP) were expressed and purified as described previously . The wild‐type sequences of human methyltransferases were as follows: SETD8 (aa 186–352), SETD7 (aa 1–366), G9a (aa 913–1193), and GLP (aa 951–1235).…”

Comparison of Molecular Recognition of Trimethyllysine and Trimethylthialysine by Epigenetic Reader Proteins

“…Starting structures were built by manually replacing the Kme3 residue of H3K4me3 with K C me3 in crystal structures of reader proteins, solvated in a 10 Å truncated octahedral box of TIP3P water [26], and neutralized explicitly with either sodium or chloride ions. AMBER12 [27] was then used to simulate the systems for 10 ns each, as previously described [11].…”

“…Atomic partial charges for each atomic center in K C me 3 correspond to those derived using the Restrained Electrostatic Potential (RESP) [36] module in AmberTools [37] calculated using HF/6-31G(d), shown in Table S3. Parameters for Kme3 were previously derived also using the RESP methodology [11].…”

“…To gain a better understanding of the exact role of lysine methylation in epigenetics, it is important to develop novel chemical tools for studying the molecular mechanisms that govern the molecular recognition of methylated lysines by reader proteins. An installation of chemically modified methylated lysine analogues into histone proteins [7] and histone peptides [8][9][10][11] has been a valuable method to study how changes in structure affect the association with reader proteins. In addition, a variety of methods have been developed to incorporate unnatural amino acids in both histone proteins and peptides, notably by auxotrophic expression systems [12] or employing the amber stop codon (TAG) [13].…”