Molecular Modeling of Interactions Regulating the Activity of the p53 Protein

该项目延续了与Ettore Appella博士实验室（LCB/NCI）的长期合作。 最初，我们专注于通过MDM 2和MDMX蛋白与N-末端反式激活结构域的结合使p53失活。 这项工作导致两种类型的竞争性抑制剂分子的发展。 第一个是基于聚N-取代甘氨酸支架的，它证明了这样的类肽可以针对蛋白质靶标进行设计的原理。 第二种更容易产生的分子是基于一种新的N-酰基多胺（NAPA）支架。 后一种分子被优化以具有与众所周知的MDM 2抑制剂Nutlin（Hoffman-La Roche）相当的结合亲和力。 然而，上级Nutlin，我们的抑制剂是有效的对MDM 2和MDMX. Secondly，我们集中在功能的相互作用p53与组蛋白乙酰转移酶共激活同源物CREB结合蛋白（CBP）和p300。 染色质结合的p53将这些蛋白质募集到基因启动子，导致组蛋白的局部乙酰化，从而导致转录所需的染色质解旋。 CBP和p300各自由以共同结构排列的七个不同结构域组成。 其中有两个转录接头锌结合结构域，Taz 1（C/H1）和Taz 2（C/H3），它们介导对转录重要的蛋白质-蛋白质相互作用。 虽然已知这两个结构域与p53的两个反式激活结构域（TAD 1和TAD 2）相互作用，但对结构细节一无所知。 在与冯汉桥博士和白亚文博士（LBMB/NCI）的合作中，我们首次阐明了p53的TAD 1与p300的Taz 2结构域相互作用的结构。 在复合物中，p53肽形成短螺旋，并通过延伸的表面与Taz 2结构域相互作用。 螺旋结合的具体方式与在与其他蛋白质的复合物中观察到的不同，最明显的是MDM 2和MDMX。 虽然复合物主要通过疏水键稳定，但静电相互作用也起作用。 我们涉及NMR、突变和热力学的其他研究表明，复合物的结构如何发生变化，并在p53的Ser 15和Thr 18残基磷酸化后进一步稳定，这被称为CBP和p300募集的翻译后修饰信号。 通过揭示p53的磷酸化残基与Taz 2的近端精氨酸残基的特异性相互作用，我们能够解释这一重要信号通路的结构基础。 这一点特别有趣，因为与第一个不同，这种相互作用不会因p53序列中类似的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正在努力追求。 一个是评估一个假定的稳定α-螺旋在C-末端调节结构域的形成，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位点特异性化学修饰的动力学，以及在不同类型的细胞应激后产生的一系列分子相互作用。

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.