Volume 12, Issue 2, Pages 461-473 (August 2003) Structural Basis for Histone and Phosphohistone Binding by the GCN5 Histone Acetyltransferase  Adrienne Clements, Arienne N Poux, Wan-Sheng Lo, Lorraine Pillus, Shelley L Berger, Ronen Marmorstein  Molecular Cell  Volume 12, Issue 2, Pages 461-473 (August 2003) DOI: 10.1016/S1097-2765(03)00288-0

Figure 1 Structure of the tGCN5/CoA/H3p19 Complex (A) Overall structure. The tGCN5 protein, CoA, and H3p19 from the ternary complex are shown in blue, purple, and green, respectively. For comparison, the H3p11 peptide from the tGCN5/CoA/H3p11 crystal structure is superimposed (using the core domain) onto the current ternary complex. Residues labeled with parentheses represent amino acids that are not modeled in their entirety due to disorder. (B) SigmaA-weighted Fo−Fc omit map. The electron density map (contoured at 2.1 σ) was generated by omitting residues within a 3.5 Å radius of H3 serine 10 followed by simulated annealing dynamics refinement at a temperature of 1000 K. (C) The H3 peptide N termini. The tGCN5 blue residues represent amino acids that contact the same residues in both histone H3 peptides. The tGCN5 aqua residues represent amino acids that are contacting additional residues in H3p19, and the tGCN5 magenta residue represents an amino acid that only interacts with H3p11 and not with any residues in H3p19. Hydrogen bonding is represented only for H3p19 by green dashed lines. The residue shown in purple is a tGCN5 arginine from a symmetry-related molecule that is interacting with tGCN5 in close proximity with the peptide. (D) The H3 peptide C termini. (E) Summary of H3 peptide-tGCN5 interactions. The gray regions of the peptide represent side chains/residues that are modeled in the H3p19 peptide and were not modeled for the H3p11 peptide. The bold bonds are shown for residues that have an increased buried surface area of at least 50 Å2 with tGCN5/CoA, when compared to tGCN5/CoA/H3p11 interactions. The solid and dashed arrows represent tGCN5 residues that are within H-bonding distance and van der Waals packing distance of the peptide, respectively. The color coding is described in the legend to (C). Strictly conserved residues among the GCN5/PCAF family are labeled with a C. Mutated residues that have no phenotype are labeled with an N. Mutated residues that affect HAT activity in the context of single and triple alanine mutation are labeled with m and M, respectively. Single mutations that affect phospho-serine-enhanced acetylation are labeled with a P. Molecular Cell 2003 12, 461-473DOI: (10.1016/S1097-2765(03)00288-0)

Figure 2 Structure of the tGCN5/CoA/H3p19Pi Complex (A) Enzymatic analysis of tGCN5 with H3p19 and H3p19Pi. Double reciprocal plot of HAT activity, showing 1/velocity versus 1/[substrate] for H3p19 (♦, R2 = 0.939) and H3p19Pi (■, R2 = 0.996). The KM and kcat values, calculated from initial velocity determination, were 796 μM and 5.6 min−1 for H3p19, and 269 μM and 1.4 min−1 for H3p19Pi, respectively. Linear fits for velocity determinations (done in triplicate) typically had R2 values greater than 0.950. (B) Overall structure. The tGCN5 protein, CoA, and the H3p19Pi peptide from the ternary complex are shown in blue, purple, and red, respectively. Both conformations of the phosphorylated serine (S10PA and S10PB) are in brown and gold, respectively. For comparison, the nonphosphorylated H3p19 peptide (green) is superimposed onto the current ternary complex. (C) SigmaA-weighted Fo−Fc omit map. The electron density map (contoured at 1.5 σ) was generated by omitting residues within 3.5 Å around both conformers of H3p19Pi phospho-serine 10. Molecular Cell 2003 12, 461-473DOI: (10.1016/S1097-2765(03)00288-0)

Figure 3 Comparison between the tGCN5/CoA/H3p19 and tGCN5/CoA/H3p19Pi Structures (A) The H3p19Pi peptide N termini. The tGCN5 blue residues represent amino acids that contact the same residues in both peptides. The tGCN5 aqua residues represent amino acids that are contacting additional residues in H3p19Pi. The tGCN5 dark green residues represent amino acids that interact with H3p19 and not with any residues in H3p19Pi. For simplicity, hydrogen-bonding interactions between H3p19Pi and tGCN5 are not shown in this figure. The residue shown in purple is a tGCN5 arginine from a symmetry-related molecule that is interacting with tGCN5 in close proximity with the peptide. (B) The H3p19Pi-peptide C termini. Hydrogen bonding between H3p19Pi and tGCN5 is represented by red dashed lines. Waters are represented as yellow spheres. (C and D) Hydrogen bonding interactions between phospho-Ser10 and tGCN5 in both conformations. The tGCN5 blue residues represent amino acids that contact Ser10 of H3p19Pi, and hydrogen bonding is represented by dashed lines. (E) Summary of the H3p19 and H3p19Pi-derived peptide-tGCN5 interactions. The gray regions of the peptide represent side chains/residues modeled in the H3p19Pi peptide and not modeled for the H3p19 peptide. The phosphate of S10P is in red. The bold bonds are shown for residues that have an increased buried surface area of at least 50 Å2 with tGCN5/CoA when compared to the tGCN5/CoA/H3p19 interactions. The solid and dashed arrows represent tGCN5 residues that are within H-bonding and van der Waals packing distance of the peptide, respectively. Residues in blue represent amino acids that contact the same residues in both peptides. Residues in aqua represent amino acids that are contacting residues only in H3p19Pi. The tGCN5 brown residues represent amino acids that only contact S10PA. Residues in green interact with H3p19 only. The tGCN5 salmon-colored residues represent amino acids that only contact S10PB. Symbols representing residue conservation and mutational sensitivity are the same as described in the legend to Figure 1E. Molecular Cell 2003 12, 461-473DOI: (10.1016/S1097-2765(03)00288-0)

Figure 4 In Vivo Effects of T11A Mutation on yGcn5-Mediated Transcription (A) Effect of yGcn5 and histone H3 mutations at the INO1 and the PCL2 promoters. The left panels summarize experiments in which yeast strains containing wild-type yGcn5, an R164A mutant of yGcn5 (corresponds to Arg113 in tGCN5), or no yGcn5 (gcn5−) are tested for expression of the INO1 gene (top panels) and the PCL2 gene (bottom panels). The right panels summarize experiments in which yeast strains containing wild-type histone H3 or an H3 mutant containing a K14A, S10A, or T11A substitution are tested for expression of the INO1 gene and the PCL2 gene. The tRNA panel was used to normalize the mRNA panel. Quantitation was performed using PhosphorImager analysis. The experiments were repeated at least two times with comparable results. (B) Effect of histone H3 mutations on Ser10 phosphorylation in bulk chromatin. Western blot analysis of bulk chromatin (upper panel) was performed using an antibody specific to phospho-Ser10 on histone H3. These experiments were performed with chromatin derived from yeast strains containing either wild-type histone H3 or H3 containing a K14A, a T11A, or a S10A substitution. Ponceau S staining of total histones (lower panel) was done on the same filter as the Western blot and the level of Ser10 phosphorylation signal was normalized to histone H3. Quantitation was done using an AlphaImager system. Molecular Cell 2003 12, 461-473DOI: (10.1016/S1097-2765(03)00288-0)

Figure 5 Summary of tGCN5/CoA/H3 Interactions (A) General features of the tGCN5/CoA/H3p19 complex and tGCN5/CoA/H3p19Pi complex phosphorylated at serine 10. The tGCN5 HAT domain is shown in blue, the H3p19 peptide is shown in green, and the H3p19Pi peptide is shown in red. Disordered regions are shown as dashed lines. Ser10, phospho-Ser10, and Thr11 are represented with a rectangle (■), a triangle (▴), and a circle (•), respectively. The tGCN5 residues and corresponding domains/secondary structure elements that interact with the N-terminal regions of the histone-derived peptides (residues 7–11 and residues 5–11 for nonphosphorylated H3 and phosphorylated H3, respectively) are shown. The tGCN5 residues that specifically contact either Ser10 or Thr11 of histone H3 are shown in orange. The residues in white only contact other H3 residues from the N terminus of the peptides through Thr11. Additionally, the tGCN5 residues (within the α1-β2 loop) that interact with the H3 residues at the C terminus of the phosphorylated H3-derived peptide (amino acids 22–23) are shown in purple. (B) Residues of H3-derived peptides that make interactions with tGCN5/CoA. For the unmodified histone H3 peptide, residues in bold type represent amino acids that have enhanced van der Waals interactions with tGCN5/CoA relative to the tGCN5/CoA/H3p11 complex. Residues in aqua represent additional hydrogen bonds in the H3p19 complex. For the phosphorylated histone H3 peptide, residues in bold type represent amino acids that have enhanced van der Waals interactions with tGCN5/CoA relative to the tGCN5/CoA/H3p19 complex. Residues in aqua represent additional hydrogen bonds relative to the tGCN5/CoA/H3p19 complex. Molecular Cell 2003 12, 461-473DOI: (10.1016/S1097-2765(03)00288-0)

Figure 6 Sequence Comparisons of GCN5/PCAF Substrates aSubstrate specificity ratios are calculated relative to H3p1935. Substrate lysines for the sequences are identified by: bGrant et al., 1999; cKuo et al., 1996; dLiu et al., 1999; Sakaguchi et al., 1998; eMunshi et al., 1998; fSartorelli et al., 1999; gKiernan et al., 1999. Molecular Cell 2003 12, 461-473DOI: (10.1016/S1097-2765(03)00288-0)