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...
p18INK4c is a member of a family of INK4 proteins that function to arrest the G1 to S cell cycle transition by inhibiting the activity of the cyclin-dependent kinases 4 and 6. The X-ray crystal structure of the human p18INK4c protein to a resolution of 1.95 A reveals an elongated molecule comprised of five contiguous 32- or 33-residue ankyrin-like repeat units. Each ankyrin-like repeat contains a beta-strand helix-turn-helix extended strand beta-strand motif that associates with neighboring motifs through beta-sheet, and helical bundle interactions. Conserved ankyrin-like repeat residues function to facilitate the ankyrin repeat fold and the tertiary interactions between neighboring repeat units. A large percentage of residues that are conserved among INK4 proteins and that map to positions of tumor-derived p16INK4 mutations play important roles in protein stability. A subset of these residues suggest an INK4 binding surface for the cyclin-dependent kinases 4 and 6. This surface is centered around a region that shows structural features uncharacteristic of ankyrin-like repeat units.
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