Molecular Modeling of Interactions Regulating the Activity of the p53 Protein

调节 p53 蛋白活性的相互作用的分子模型

• 批准号: 10487233
• 负责人: Stewart Durell
• 金额: $ 21.66万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10487233
• 关键词: Affinity; Apoptosis; Architecture; Arginine; Binding; Biochemical Pathway; C-terminal; CREBBP gene; Cancer Biology; Cell Cycle Arrest; Cell Cycle Kinetics; Cellular Stress; Chemicals; Chromatin; Collaborations; Complex; DNA Damage; Development; EP300 gene; Electrostatics; Genes; Genetic Transcription; Histone Acetylation; Hydrophobicity; Laboratories; Lead; MDM2 gene; Mediating; Modification; Monitor; Mutation; N-substituted Glycines; N-terminal; Normal Cell; Pattern; Peptides; Peptoids; Phosphorylation; Play; Post-Translational Protein Processing; Proteins; Role; Series; Serine; Signal Pathway; Signal Transduction; Site; Structure; Surface; TP53 gene; Thermodynamics; Threonine; Transactivation; Work; Zinc; alpha helix; base; design; drug development; histone acetyltransferase; inhibitor/antagonist; interest; molecular modeling; molecular targeted therapies; novel; promoter; protein protein interaction; recruit; response; scaffold; structural biology; transcription factor

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. Recently we finished refining and analyzing the NMR structure of the p300-Taz2/p53-TAD2 complex.

该项目继续与Ettore Appella博士（LCB/NCI）的实验室进行了长期的合作。最初，我们通过MDM2和MDMX蛋白与N末端的反式激活结构域的结合将p53灭活。这项工作导致了两种类型的竞争抑制剂分子的发展。第一个基于聚氮基取代的甘氨酸支架，是原理证明这种肽可以针对蛋白质靶标设计的。第二个更容易产生的分子是基于一种新型的N-酰丙胺（NAPA）支架。后一种分子被优化为具有与众所周知的MDM2抑制剂Nutlin（Hoffman-La Roche）相当的结合亲和力。但是，我们的抑制剂优于Nutlin，对MDM2和MDMX都有效。随后，我们集中在p53与组蛋白乙酰转移酶共激活酶同源物CREB结合蛋白（CBP）和p300的功能相互作用上。染色质结合的p53将这些蛋白质募集到基因启动子中，从而导致组蛋白的局部化乙酰化，因此需要放松转录所需的染色质。 CBP和P300各自由在公共结构中排列的七个不同的域组成。其中包括两个转录适配器锌结合域，TAZ1（C/H1）和TAZ2（C/H3），它们介导对转录很重要的蛋白质 - 蛋白质相互作用。尽管已知这两个域都与p53（TAD1和TAD2）的反式激活结构域相互作用，但对结构细节一无所知。与Drs合作。 Hanqiao Feng和Yawen Bai（LBMB/NCI），我们是第一个阐明p53与p300 TAZ2域的TAD1相互作用结构的人。在复合物中，p53肽形成一个短螺旋，并通过延伸表面与TAZ2结构域相互作用。螺旋结合的特定方式与与其他蛋白质的复合物中观察到的不同方式，最著名的是使用MDM2和MDMX。尽管该复合物主要由疏水键稳定，但静电相互作用也起作用。我们的其他涉及NMR，突变和热力学的研究表明，在残基Ser15和Thr18处p53的磷酸化后，复合物的结构如何移动并进一步稳定，这被称为募集CBP和P300的翻译后修饰信号。通过揭示p53的磷酸化残基与TAZ2近端精氨酸残基的特异性相互作用，我们能够解释此重要信号通路的结构基础。当前，我们正在追求与p53的第二个反式激活结构域TAD2的p300 TAZ2结构域的结构。这是特别感兴趣的，因为与第一个不同，相互作用不会因p53序列中类似丝氨酸和苏氨酸残基的磷酸化而改变。最后，我们至少有两个新的方向可以追求。一种是评估C末端调节域中推定的稳定α-螺旋的形成，并确定修饰对p53四聚体稳定的影响。另一种是直接在细胞中监测p53的位点特异性化学修饰的动力学以及遵循不同类型的细胞应激的一系列分子相互作用。最近，我们完成了P300-TAZ2/p53-TAD2复合物的NMR结构的完善和分析。