Structure of Tetrahymena GCN5 bound to coenzyme A and a histone H3 peptide

Abstract

Gene activation is a highly regulated process that requires the coordinated action of proteins to relieve chromatin repression and to promote transcriptional activation. Nuclear histone acetyltransferase (HAT) enzymes provide a mechanistic link between chromatin destabilization and gene activation by acetylating the epsilon-amino group of specific lysine residues within the aminoterminal tails of core histones to facilitate access to DNA by transcriptional activators. Here we report the high-resolution crystal structure of the HAT domain of Tetrahymena GCN5 (tGCN5) bound with both its physiologically relevant ligands, coenzyme A (CoA) and a histone H3 peptide, and the structures of nascent tGCN5 and a tGCN5/acetyl-CoA complex. Our structural data reveal histone-binding specificity for a random-coil structure containing a G-K-X-P recognition sequence, and show that CoA is essential for reorienting the enzyme for histone binding. Catalysis appears to involve water-mediated proton extraction from the substrate lysine by a glutamic acid general base and a backbone amide that stabilizes the transition-state reaction intermediate. Comparison with related N-acetyltransferases indicates a conserved structural framework for CoA binding and catalysis, and structural variability in regions associated with substrate-specific binding.

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).…”

Molecular basis of the acetyltransferase activity of MEC-17 towards α-tubulin

“…As in the Gcn5/acetyl-CoA [6] and PCAF/CoA complexes, the cofactor assumes a sharply bent conformation and the interactions between the cofactor and DrMEC-17 are largely conserved (Supplementary information, Figures S1, S2 and S3, and Data S1). Mutations of some involved residues, including Arg126, Gly128, Gly130 and Ser154, resulted in significant decreases in the enzyme activity ( Figure 1D).…”

“…On the other hand, the side chain of Gln53 points toward the substrate-binding cleft (Supplementary information, Figure S2, left panel). In the structure of the Gcn5/CoA/H3 peptide complex, several hydrophobic residues at this region that bind to CoA also interact with the substrate peptide [6]. Previously, the Chalfie group reported two missense mutations in allele of C. elegans MEC-17, u265, one of which (Leu46) corresponds to Gln53 of DrMEC-17 [9].…”

“…In the Gcn5 structures, the cofactor is located in a small cleft and the substrate peptide is bound in a big cleft formed by the N-and C-terminal segments and the antiparallel β3-β4 sheet of the core region [6,7,11]. In the DrMEC-17/acetyl-CoA complex, the cofactor is bound with a similar mode (Supplementary information, Figure S2) and a presumed substrate-binding cleft is also detected at an equivalent position ( Figure 1A, right Wenjing Li et al 1709 npg panel), although significant differences in the size and electrostatic properties are observed.…”

“…On the other hand, the C-terminal segment of MEC-17 displays substantial divergence compared with Gcn5 ( Figure 1A, right panel), and there is no structural equivalent of the α5-β6 loop of Gcn5 that has been shown to be important for the histone binding [6,11]. Gcn5 functions in large protein complexes and two regions, one flanking the histone-binding cleft and the other at the C-terminal surface, have been proposed to interact with other proteins to modulate the acetyltransferase activity of Gcn5.…”