Structural studies of selected H1 N-terminal domains

Abstract: DNA inside eukaryotic nuclei is highly packaged with histones and non-histone proteins in a complex structure called „chromatin‟. The basic repeating unit of chromatin is the nucleosome. The nucleosome core particle is composed of ~147 bp of DNA wrapped around an octamer of two molecules each of the core histones H2A, H2B, H3 and H4. A fifth histone, the linker histone (H1/H5), binds to the nucleosome and further condenses it into the chromatin higher-order structure, thereby regulating gene expression, and other cellular processes. In mammals, there are 11 variants of H1, which differ in patterns of expression, chromosomal distribution and chromatin binding dynamics. The canonical linker histone has a well-structured central globular domain, flanked by unordered N- and C-terminal tail domains. The globular and C-terminal domains have been extensively studied, but the role of the N-terminal domain (NTD) remains unclear. Understanding the structure of the NTDs of H1 variants may contribute to understanding the differential roles of H1 variants in chromatin structure and dynamics. In order to investigate the structure of the NTDs of H1x and H1.4, fusion proteins containing these NTDs fused to the globular domain of H5 were expressed in E. coli and purified by FPLC. Chromatosome protection assays and limited tryptic proteolysis were used to confirm the functionality and structural integrity of the recombinant proteins prior to structural studies. Circular dichroism (CD) analysis showed that the NTDs had few structured elements in „low salt‟ (10 mM sodium phosphate, pH 7.0), but that structural elements were induced in the presence of a helix stabilizer (1 M sodium perchlorate). In order to predict the tertiary structure of the NTDs and to gain further insight into possible binding partners and binding modes, molecular dynamics simulations were employed. These simulations further suggested the presence of distinct structures in NTDs under both „low‟ and „high salt‟ conditions. FATCAT structural alignment revealed homologues of predicted NTD structures in the PDB, most of which were involved in DNA binding. Homology-based DNA-protein docking suggested that NH1.4 binds the major groove of the DNA double helix in a manner similar to the homeodomain, while NH1x binds the minor and major groove of the DNA in a manner similar to the homeobox DNA-binding motifs. Our results cement the view of H1 NTDs as bona fide intrinsically unstructured proteins, and suggest that the NTDs may interact with DNA through distinct binding modes.M.Sc. (Biochemistry