Molecular Basis for Chaperone Control of Rtt109 Acetylation of Histone H3-H4




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Acetylation is one of many protein post-translational modifications (PTMs) that frequently occurs in the cell. One type of acetylation is when the acetyl group from acetyl-coenzyme A (Ac-CoA) is transferred onto the ε-amino group of lysine sidechains. Histones are highly basic proteins that associate with genomic DNA and compact it into chromatin in the nucleus of the cell. They are often accompanied by a group of histone-binding proteins called histone chaperones in events such as nucleosome assembly/disassembly, histone transport and nuclear import. Being rich in lysine content, histones are frequently acetylated and therefore influence chromatin structure. Enzymes that carry out histone acetylation are termed histone acetyltransferases (HATs). Rtt109 is one such HAT that is found in fungal species, and requires association with histone chaperones to efficiently acetylate histones. Vps75 and Asf1 are the two known histone chaperones that when bound to Rtt109, enhance its enzymatic activity significantly. They also play a role determining Rtt109 selectivity and specificity towards different lysine residues in histones H3-H4. Cells deleted for Rtt109, Asf1 or both, are highly sensitive to genotoxic exposure; and it has been shown that Rtt109 acetylation of K56 is required for a cell to maintain its genomic integrity. This has made Rtt109 and its chaperone-containing complexes attractive anti-fungal therapeutic targets. The structure, dynamics and function of Rtt109 complexes are the focus of this dissertation. Utilizing comprehensive biophysical and biochemical analysis, we first investigate molecular interactions between Vps75 and H3-H4. We report the stoichiometry of binding in multiple ionic conditions and compare their interactions to a homologous complex containing Nap1. We identify the interface between Vps75 and H3-H4, and reveal how specific structural elements are tailored for Vps75 chaperoning activity with Rtt109. Next, we add Rtt109 to the Vps75-(H3-H4) complex and extensively characterize complex homogeneity and absolute stoichiometry. We define a detailed step-by-step Rtt109-Vps75 co-expression and purification protocol that maximizes yield and purity. We show the stoichiometry of binding is 1:2, with a second Rtt109 binding only at high concentration and readily replaced with H3-H4. We show that Rtt109-Vps75-(H3-H4) has a 1:2:1 unit that can self-associate to become a 2:4:2 complex through the H3-H3 contacts in a H3-H4 tetramer. Our large-scale reconstitution methods for various Rtt109 complexes paved the way for acquiring high-resolution structures via crystallography or cryo-electron microscopy. It also facilitated solution characterization via hydrogen-deuterium exchange mass spectrometry (HDXMS). Finally, we added Asf1, the last binding partner to reconstitute the double-chaperone complex − Rtt109-Vps75-(H3-H4)-Asf1. This allows for a comprehensive comparative HDX-MS experiment to uncover the mechanism behind chaperone activation of Rtt109. We purified and reconstituted eleven relevant protein complexes for analysis. We identify direct binding sites between each member of the complex and compare them to existing structures, and show different conformations upon addition of each chaperone. These results elucidate the acetylation mechanisms facilitated by cross-talk between two histone chaperones Vps75 and Asf1.



Chemistry, Biochemistry