The structural studies of bromodomain proteins in complex with histone crotonylation peptides

Abstract

Histone modifications are the key epigenetic regulations that play important roles in various cellular activities. Histone lysine crotonylation (Kcr), firstly discovered in 2011, are evolutionary conserved for a wide range of eukaryotic species. Histone Kcr were found to mainly associate with gene promoters and enhancers and stimulate gene expression. Bromodomains are protein interaction modules that were known as acetylation readers. Our lab discovered that bromodomains could also bind to histone crotonylation peptides. According to the microarray results, bromodomain proteins that have strongest binding with crotonylation peptides, PB1brd6 with H3K18cr and BRD4brd2 with H3K4cr or H3K9cr were chosen to further study their binding patterns through X-ray crystallography. Co-crystallization of PB1¬brd6 (773-914 a.a.) with or without his-tag with H3K18cr was tried, but only apo structures of PB1brd6 were solved. And BRD4brd2 truncated to 333-460 a.a. or 347-460 a.a with his tag or his-SUMO tag were overexpressed, purified. And then BRD4brd2 with or without tags were used to get co-crystals. Finally, the co-crystal structure of BRD4brd2 with H3K9cr at the resolution of 3.4 Å and apo structures of BRD4brd2 were solved. ITC tests were done to measure the interactions between the two bromodomain proteins and the three histone crotonylation peptides. However, the results did not show a standard ‘S’ curve. In-silico docking results showed it is possible that PB1brd6 binds to H3K18cr. In summary, the work I did is a preliminary support that bromodomain proteins could recognize histone crotonylation, which will lead to further investigation on the interactions between different bromodomain proteins and crotonylated peptides, thus revealing the underlying mechanisms and discovering new therapeutic methods of relevant diseases.