Sequence alignment of the GCN5 family of HAT domains. The primary sequence of the yeast GCN5 (yGCN5) HAT domain used for the structure determination is shown at the top of the alignment. Sequences from the homologous HAT domains from GCN5 of Arabidopsis, Drosophila, human, and Tetrahymena as well as with human P/CAF are aligned [clustal program (http://www-igbmc.u-strasbg.fr/BioInfo/ClustalW/)] and displayed [boxshade program (http://www.ch.embnet.org/software/BOX_form.html)]. Black and gray backgrounds are used to indicate identical and/or conserved residues found in at least 50% of the proteins at a given position, respectively. Secondary structural elements within the HAT domain of yGCN5 are shown above the sequence alignment. ●, residues that are buried within the core of the protein; A, residues that are implicated from the HAT1 and SmAAT structures to contact the CoA cofactor (53); ▵, solvent exposed residues that are presumably accessible for coenzyme or substrate binding and catalysis; ∗, the position of the putative general base (Glu173) for histone acetylation. Positions of alanine mutations that decrease HAT activity are indicated below the sequence alignment: Triple mutations are indicated with gray bars (38), and single mutations are indicated with black bars (37). Triple alanine mutations that have negligible effect on HAT activity are indicated with open rectangles (all of the single mutations are contained within the triple mutations). The GNAT conserved motifs D, A, and B (39) are indicated below the sequence alignment, with black shading indicating regions of high sequence homology within the GNAT superfamily.

Functional defects of the yGCN5 E173Q mutant and the putative role of Glu173 in catalysis. (A) Growth of yeast cells harboring empty vector (gcn5Δ), wild-type GCN5 plasmid, or GCN5 plasmid with a Glu-to-Gln mutation at residue 173 (E173Q). Strains were streaked on a minimal media plate (Left) or were grown in liquid minimal media at 30°C for determination of log-phase generation (doubling) times of the cells (Right). (B) Transcriptional activation in vivo. Shown are the activities of extracts from cells containing activator plasmid GAL4-VP16_FA or LexA-GCN4 and the appropriate β-galactosidase reporter plasmid. (C) HAT activity assays in vitro. Purified yGCN5 and yGCN5-E173Q HAT domain proteins (0.1 μg) were used for liquid HAT assays with ³H-AcCoA and free histone substrates at pH 7.5 and 25°C.

Structure of yGCN5 and related enzymes. (a) Structure of the yGCN5 HAT domain. The four subdomains of the protein are color-coded; the structurally conserved subdomain that makes up the core (motifs A and D) is colored blue, motif B is colored aqua, and the N-terminal and C-terminal flanking regions are colored red and green, respectively. (b) Structure of residues 130–320 (C terminus) of HAT1 with the same orientation and color coding as in a. The AcCoA cofactor is shown in magenta. (c) Structure of SmAAT with the same orientation and color coding as in a. The CoA cofactor is shown in magenta. (d) Structure of residues 80–260 (N terminus) of NMT with the same orientation and color coding as in a.

Implications of the yGCN5 structure for HAT function. (a) Strictly conserved residues within the GCN5 subfamily of acetyltransferases (Fig. 2) that are buried and thus play a role in protein stability are indicated as red balls, and strictly conserved and solvent exposed yGCN5 residues (Fig. 2) (implicated for histone substrate or CoA cofactor binding or catalysis) are shown as green side-chains. AcCoA from the HAT1 structure is placed in the cofactor binding site for reference by superposing the HAT1 core domain. Secondary structural elements are indicated for reference. (b) Mutations in yGCN5 that decrease HAT activity are mapped onto a schematic representation of the yGCN5 HAT domain. Triple mutations are shown as green side chains, and single mutations are shown as red side chains. (c) Electrostatic surface of the yGCN5 HAT domain. Red indicates acidic regions, and blue indicates basic regions. A backbone trace of the protein is superimposed in green. (d) SigmaA-weighted |F_o| − |F_c| omit map around the region of residue Glu173, which is implicated to function as a catalytic base for substrate catalysis. The map was generated by omitting residues within an 8-Å radius of Glu 173 followed by simulated annealing dynamics refinement at a temperature of 1,000 K. The map is contoured at 1.8 sigma. The green sphere indicates a water molecule.