Crystal structure and mechanism of histone acetylation of the yeast GCN5 transcriptional coactivator

Abstract

The yeast GCN5 (yGCN5) transcriptional coactivator functions as a histone acetyltransferase (HAT) to promote transcriptional activation. Here, we present the high resolution crystal structure of the HAT domain of yGCN5 and probe the functional importance of a conserved glutamate residue. The structure reveals a central protein core associated with AcCoA binding that appears to be structurally conserved among a superfamily of N-acetyltransferases, including yeast histone acetyltransferase 1 and Serratia marcescens aminoglycoside 3-Nacetyltransferase. A pronounced cleft lying above this core, and flanked by N-and C-terminal regions that show no sequence conservation within N-acetyltransferase enzymes, is implicated by cross-species conservation and mutagenesis studies to be a site for histone substrate binding and catalysis. Located at the bottom of this cleft is a conserved glutamate residue (E173) that is in position to play an important catalytic role in histone acetylation. Functional analysis of an E173Q mutant yGCN5 protein implicates glutamate 173 to function as a general base for catalysis. Together, a correlation of the yGCN5 structure with functionally debilitating yGCN5 mutations provides a paradigm for understanding the structure͞function relationships of the growing number of transcriptional regulators that function as histone acetyltransferase enzymes.Gene activation is a tightly regulated process that relies on the coordinated activities of several different proteins. Sequencespecific transcriptional activators play an important role in nucleating gene expression by recruiting the basal transcriptional machinery, which includes TATA box-binding proteins, TAFs (TATA box-binding protein-associated proteins), polymerase, and other protein cofactors, to DNA (1, 2). Under physiological conditions, the transcriptional machinery has to deal with a DNA template that is complexed with histones that form nucleosomes and that are assembled into higher order chromatin (3). Because chromatin assembly strongly represses transcription (4, 5), DNA regulatory proteins must physically destabilize nucleosome͞DNA interactions to facilitate transcriptional activation.The relatively recent findings that histone acetyltransferase (HAT) and histone deacetyltransferase enzymes are proteins that had been previously characterized as transcriptional cofactors has revealed a direct mechanistic link between chromatin modification and transcriptional regulation (6-10). Specifically, a number of transcriptional cofactors with HAT activity have been identified, including GCN5 .Of the HAT enzymes identified to date, yGCN5 is currently the best characterized. Recombinant GCN5 has been shown to efficiently acetylate free histones with a strong preference for lysine 14 of histone H3 and a somewhat lower preference for lysines 8 and 16 of histone H4 (32). Interestingly, acetylation of nucleosomal histones requires that GCN5 be part of one of two distinct multiprotein complexes called Ada and SAGA (Spt-Ada-GCN5-acetyltransferase...

Acetylation of adenovirus E1A regulates binding of the transcriptional corepressor CtBP

“…The catalytic domain of P͞CAF is similar to that of Gcn5, whose structure has been solved (51). Crystallographic analysis of the Tetrahymena Gcn5͞CoA͞histone H3 complex indicated that sequences carboxyl-terminal to the target Lys are particularly critical for enzyme recognition (52).…”

Acetyl-CoA induces transcription of the key G1 cyclin CLN3 to promote entry into the cell division cycle in Saccharomyces cerevisiae

“…We assayed CLN3 transcription following rich medium repletion in a gcn5Δ or gcn5 E173Q mutant that is catalytically inactive (46). In addition, we tested the sgf73Δ mutant, which lacks an important subunit of SAGA that is dynamically acetylated in response to acetyl-CoA (36).…”

Chemistry of acetyl transfer by histone modifying enzymes: structure, mechanism and implications for effector design

“…The structure of HAT domains from several members of the Gcn5/PCAF family has been determined by X-ray crystallography and nuclear magnetic resonance, both alone and in complex with substrates (Clements et al, 1999;Lin et al, 1999;Rojas et al, 1999;Trievel et al, 1999;Yan et al, 2000Yan et al, , 2002Poux et al, 2002;Poux and Marmorstein, 2003). These structures reveal that the A, B and D regions of the GNAT proteins form a central core that is structurally homologous to other GNAT proteins and mediates conserved Ac-CoA interactions ( Figure 1a).…”

“…The structure of the nascent yeast Gcn5 HAT domain, together with mutational and enzymatic studies, reveals that the Gcn5/PCAF HAT enzymes use a ternary complex mechanism (Tanner et al, 1999;Trievel et al, 1999) involving deprotonation of the N-e-nitrogen of the target lysine by a conserved glutamate residue (E173 in yeast Gcn5) within the core domain followed by direct nucleophilic attack of the deprotonated nitrogen on the acetyl group of the Ac-CoA bound cofactor (Figures 1c and d). Initial structural studies on the yeast Esa1 HAT domain, together with mutational and in vitro and in vivo studies, revealed that a glutamate residue (E338 in yeast Esa1) that showed structural alignment with the corresponding catalytic glutamate residue of Gcn5/ PCAF played a similar catalytic role.…”