Mechanistic and structural studies on p300/CBP transcriptional coactivator

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.;We have also explored the mechanisms of p300 autoacetylation by kinetic and mass spectrometric analysis, based on the prior discovery of an autoacetylated regulatory loop in the p300 HAT domain. We deduced that autoacetylation occurs primarily through an intermolecular mechanism. Kinetic analysis of the 17 acetylation sites observed reveals a class of rapid events followed by a slower set of modifications.;In addition, we have studied the transcription factor ATF2 and the properties of a novel CoA probe and their interactions with p300. These experiments have revealed new insights into the structure, regulation, and function of p300/CBP.