title: The role of loop ZA and Pro371 in the function of yeast Gcn5p bromodomain revealed through molecular dynamics and experiment.
abstract: Biological experiments were combined with molecular dynamics simulations to understand the importance of amino acidic residues present in the bromodomain of the yeast histone acetyltransferase Gcn5p. It was found that residue Pro371 plays an important role in the molecular recognition of the acetylated histone H4 tail by Gcn5p bromodomain. Crystallographic analysis of the complex showed that this residue does not directly interact with the histone substrate. It has been demonstrated that a double mutation Pro371Thr and Met372Ala in the Gcn5p bromodomain impairs chromatin remodeling activity. It is demonstrated here that, in this double mutant and in the fully deleted bromodomain strain, there is lower growth under amino acid deprivation conditions. By in vitro surface plasmon resonance (Biacore) experiments it is shown that the binding affinity of the double mutation to acetyl lysine 16 histone H4 peptide decreases. Molecular dynamics simulations were used to explain this loss in acetyl lysine-Gcn5p bromodomain affinity, in the double mutant. By comparing nanosecond molecular dynamics trajectories of the native as well as the single and doubly mutated bromodomain, it is concluded that the presence of Pro371 is important to the functionality of the Gcn5p bromodomain. In the simulation a point mutation involving this highly conserved residue induced an increase in the flexibility of the ZA loop, which in turn modulated the exposure of the binding pocket to the acetyl lysine. The combined double mutations (Pro371Thr-Met372Ala) not only markedly perturb the motion of the ZA loop but also destabilize the entire structure of the bromodomain.
authors.affiliation: Commissariat à l'Energie Atomique, DSV-DBJC-SBFM, Centre d'Etudes Saclay, 91191 Gif-sur-Yvette Cedex, France. Francesco.Pizzitutti@roma1.infn.it
authors.forename: Francesco
authors.initials: F
authors.lastname: Pizzitutti
authors.forename: Andrea
authors.initials: A
authors.lastname: Giansanti
authors.forename: Paola
authors.initials: P
authors.lastname: Ballario
authors.forename: Prisca
authors.initials: P
authors.lastname: Ornaghi
authors.forename: Paola
authors.initials: P
authors.lastname: Torreri
authors.forename: Giovanni
authors.initials: G
authors.lastname: Ciccotti
authors.forename: Patrizia
authors.initials: P
authors.lastname: Filetici
mesh_terms: D000107:Acetylation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D051548:Histone Acetyltransferases / Q000737:chemistry* / Q000378:metabolism*; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D000099224:p300-CBP-Associated Factor
chemical_list: D051548:Histone Acetyltransferases; D006657:Histones; D011392:Proline; D029701:Saccharomyces cerevisiae Proteins; C501392:GCN5 protein, S cerevisiae; D000099224:p300-CBP-Associated Factor