| The genetic information of eukaryotic organism exists in the chromatin. The nucleosomes as chromatin basic repeating units are comprised of core histones, linker histone HI and genomic DNA.The nucleosome core consists of one H3-H4 tetramer and two H2A-H2B dimers that are wrapped around by 147 bp DNA. Histone chaperones are critical for regulating post-transcriptional modifications of histones and nucleosome (dis)assembled is also highly regulated by various histone chaperones. Hifl has been reported to be a component of nuclear HAT-B complex, which specifically acetylates Lys5 and Lys12 of nuclear histone H4. Histone acetyltransferase HAT-B complex comprises three components:HAT1, HAT2 and Hifl. HAT1, which play a role in acetylation the amino acid residues; HAT2 assists HAT1 to acetylate the amino acid residues. It seems that Hifl has no significant contribution to the enzyme activity of the HAT-B complex., but Hifl as histone chaperone interacts with H3-H4. Deletion of Hifl results in a defect in telomeric silencing and DNA double-strand break repair. However, how hifl binds to histones not yet reported.Here, we solved the complex structure of Hifl binding to H2A-H2B dimer complex. This structure reveal that two Hifl molecules flank one H2A-H2B dimer with two distinct patterns. One pattern is TPR extended binding, another pattern is nucleosomal DNA competitive binding. We have also employed ITC and SPR to validate the mechanism of Hifl binding with H2A-H2B. According to the GST-pull down assay, Hifl binds to H2A-H2B dimer in low ionic strength conditions, but binds to H3-H4 even under high ionic strength. Through the ITC experiments, the heats of injections from Hifl titrating into H2A-H2B dimer fit well with the binding model of two sets of sites, but the binding heat between Hifl and the H3-H4 tetramer was fitted well by the one set of sites model. Thus, the binding of Hifl to H2A-H2B differs significantly from the binding to the H3-H4 tetramer. Hifl has a long inserted acid loop interrupts its TPR domain, and the acid loop have prominent roles in binding to the H2A-H2B dimer and H3-H4. Acid loop binding by the H3-H4 tetramer leads to conformational changes in these patches. This may result in the release of the H2A-H2B dimer from Hifl. Human NASP is the homolog of yeast Hifl, and they are members of the SHNi-TPR protein family. We find that sNASP is similar with Hifl on binding with histone. Thus, we speculate SHNi-TPR family proteins may be very similar in structure, and participate in the same regulatory process.Hifl is a component of the HAT-B complex.We find that Hifl interact with Hat2,not Hat1.But we also need further exploration on the interaction with Hif1 and Hat, Hat2.Saccharomyces cerevisiae tRNA(m~1A58) methyltransferase(ScTRM6-TRM61) catalytics and transfers methyl from S-adenosyl-methionine(SAM) to 58 adenine of the tRNA. This methyl transferase is widely distributed in archaea, prokaryotic, eukaryotic organisms, but in archaea and prokaryotic, it forms monomers or polymers, each monomer has both SAM-binding domain and tRNA binding domain. In eukaryotes, it is composed of two subunits, namely TRM6 and TRM61. One subunit TRM61 plays a role in methyltransferase activity and the other subunit TRM6 has the fuction of binding tRNA. Through the structural alignment, we found that ScTRM61 is similar with the protein in archaea and prokaryotic, and the activity center is also highly conserved. The structure of human tRNA(m’A58) methyltransferase(hcTRM6-TRM61) has been solved, they reported the complex of tRNA and hcTRM6-TRM61. By comparison ScTRM6-TRM61 and hcTRM6-TRM61, we find some difference. This will help us to further explain the mechanism of the tRNA (m~1A58) methyltransferase. |
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