This project continues a long-standing collaboration with the Laboratory of Dr. Ettore Appella (LCB/NCI). Initially we focused on the inactivation of p53 by the binding of the MDM2 and MDMX proteins to the N-terminal, transactivation domain. This work lead to the development of two types of competitive inhibitor molecules. The first, based on a poly N-substituted glycine scaffold, was the proof of principle that such peptoids could be designed against a protein target. The second, easier to produce molecule was based on a novel, N-acylpolyamine (NAPA) scaffold. This latter molecule was optimized to have a binding affinity comparable to the well-known MDM2 inhibitor Nutlin (Hoffman-La Roche). However, superior to Nutlin, our inhibitor is potent against both MDM2 and MDMX. Subsequently, we have concentrated on the functional interactions of p53 with the histone acetyltransferase coactivator homologs CREB-binding protein (CBP) and p300. Chromatin-bound p53 recruits these proteins to the gene promoter, resulting in localized acetylation of the histones, and thus the required unwinding of the chromatin needed for transcription. CBP and p300 are each composed of seven distinct domains arranged in a common architecture. Among these are two transcriptional adaptor zinc-binding domains, Taz1 (C/H1) and Taz2 (C/H3), which mediate protein-protein interactions important for transcription. While both these domains were known to interact with both transactivation domains of p53 (TAD1 &TAD2), nothing was known of the structural details. In collaboration with Drs. Hanqiao Feng and Yawen Bai (LBMB/NCI), we were the first to elucidate the structure of the interaction of the TAD1 of p53 with the Taz2 domain of p300. In the complex, the p53 peptide forms a short helix and interacts with the Taz2 domain through an extended surface. The specific way in which the helix is bound is different from what has been observed in complexes with other proteins, most notably with MDM2 and MDMX. While the complex is primarily stabilized by hydrophobic bonds, electrostatic interactions also play a role. Our additional studies involving NMR, mutations and thermodynamics indicated how the structure of the complex shifts and is further stabilized upon phosphorylation of p53 at residues Ser15 and Thr18, which was known as post-translational modification signals for the recruitment of CBP and p300. By revealing the specific interactions of the phosphorylated residues of p53 with proximal arginine residues of Taz2 we were able to explain the structural basis for this important signaling pathway. Currently, we are pursuing the structure of the complex of the p300 Taz2 domain with TAD2, the second transactivation domain of p53. This is of particular interest, because, unlike the first, the interaction is not altered by phosphorylation of the analogous serine and threonine residues in the p53 sequence. Finally, we have at least two new directions we are gearing-up to pursue. One is to evaluate the formation of a putative stabilizing alpha-helix in the C-terminal regulatory domain, and to determine the effect of modifications on the stabilization of the p53 tetramer. The other is to monitor directly in cells the kinetics of the site-specific chemical modifications of p53 and the resultant series of molecular interactions that follow different types of cellular stresses. This last year we finished refining and analyzing the NMR structure of the p300-Taz2/p53-TAD2 complex.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Scientific Cores Intramural Research (ZIC)
Project #
1ZICBC011077-07
Application #
8938463
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Miller Jenkins, Lisa M; Feng, Hanqiao; Durell, Stewart R et al. (2015) Characterization of the p300 Taz2-p53 TAD2 complex and comparison with the p300 Taz2-p53 TAD1 complex. Biochemistry 54:2001-10
Feng, Hanqiao; Jenkins, Lisa M Miller; Durell, Stewart R et al. (2009) Structural basis for p300 Taz2-p53 TAD1 binding and modulation by phosphorylation. Structure 17:202-10
Hayashi, Ryo; Wang, Deyun; Hara, Toshiaki et al. (2009) N-acylpolyamine inhibitors of HDM2 and HDMX binding to p53. Bioorg Med Chem 17:7884-93