Mechanistic and Structural Studies on P300/CBP Transcriptional Coactivator

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ProQuest, 2009 - 206 pages
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The transcriptional coactivator paralogs p300 and CBP are among the most intensively studied proteins in the gene regulation field and are linked to a broad array of biological pathways. Dysregulation of p300/CBP HAT activity contributes to various diseases, including cancer. We have solved the X-ray crystal structure of a semisynthetic heterodimeric p300 HAT domain in complex with a bisubstrate inhibitor, Lys-CoA. The 1.7A structure demonstrates that p300/CBP is a distant cousin of other structurally characterized HATs, but reveals a number of novel structural features. Mutagenesis, product inhibition kinetics, and fluorescence binding studies all strongly support a Theorell-Chance (or "hit-and-run") mechanism; this is the first report of this mechanism for the HAT family. With the help of the structure, a number of disease-associated mutations can be readily explained. The acid surface of the proposed substrate binding cavity explains the substrate preference of basic residues for p300. These studies pave the way for drug development and novel epigenetic therapies involved with p300/CBP HAT activity.
  

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Contents

1 2 8 A Crystal Structure of the Nucleosome
2
3 Hat 1 structure
9
6 The Esal Catalytic Site
15
8 Schematic representation of the p300CBP primary structure
24
10 FoxO pathways in response to external and internal stimuli
30
12 Structure of LysCoA and H3CoA20
36
Chapter II Autoacetylation Study of p300 HAT
53
1 The domain structure of p300 HAT domain highlighted with
55
3 SDSPAGE gel 12 of different fractions after cleavage and ligation
85
7 Acetyltransferase activity of the heterodimeric p300 HAT
98
11 Circular dichroism spectra of wt p300 HAT and p300 HATAC at 42C
105
15 Structureguided mutagenesis and functional characterization
113
17 Substrate binding surface and mutagenesis studies
121
18 Schematic representation of the TheorellChance mechanism
127
20 Fluorescence binding assays of CoASH and acetonylCoA to the p300
133
23 pHrate profile of wildtype loopdeleted p300 HAT with saturating
140

2 Autoacetylation of p300 HAT domain as a function of time
62
6 Effects of acetylCoA on p300 HAT autoacetylation
68
Chapter III Structural and Mechanistic Study of p300 HAT
77
1 Partial trypsin digestion study and the proposed domain structure
79
Chapter IV ATF2 interaction affinity labeling and human disease mutations
146
1 Domain structure of ATF2 bzip domain is the DNA binding domain
147
2 Affinity binding assays with immobilized GSTATF2bZIP for different
157
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