Molecular Modeling of Interactions Regulating the Activity of the p53 Protein

该项目延续了与Ettore博士实验室的长期合作 Appella（LCB/NCI）。最初，我们关注的是通过MDM 2的结合使p53失活。 和MDMX蛋白的N-末端反式激活结构域。这项工作导致了发展 两种竞争性抑制剂分子。第一，基于聚N-取代的 甘氨酸支架，是这样的类肽可以设计成针对 蛋白质靶标第二种更容易生产的分子是基于一种新的N-酰基多胺 （国家适应行动方案）脚手架。后一种分子被优化以具有与以下分子相当的结合亲和力： 众所周知的MDM 2抑制剂Nutlin（Hoffman-La Roche）。然而，我们的上级于纳特林， 抑制剂对MDM 2和MDMX都有效。随后，我们集中讨论了 p53与组蛋白乙酰转移酶共激活因子同源物的功能相互作用 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中， 启动子，导致组蛋白的局部乙酰化，因此需要解旋 转录所需的染色质。CBP和p300分别由7个不同的 域排列在一个共同的架构。其中有两个转录衔接子 锌结合结构域，Taz 1（C/H1）和Taz 2（C/H3），介导蛋白质-蛋白质相互作用 对转录很重要。虽然这两个域都与 p53的反式激活结构域（TAD 1和TAD 2），结构细节一无所知。 与冯汉桥博士和白亚文博士（LBMB/NCI）合作，我们是第一个 阐明p53的TAD 1与p300的Taz 2结构域的相互作用的结构。在 在复合物中，p53肽形成一个短螺旋，并通过一个螺旋与Taz 2结构域相互作用。 延伸表面螺旋结合的具体方式与以往不同， 在与其他蛋白质的复合物中观察到，最明显的是与MDM 2和MDMX。虽然复杂的 主要通过疏水键稳定，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， 当p53在丝氨酸15位残基磷酸化时，复合物发生位移并进一步稳定， Thr 18是CBP募集的翻译后修饰信号 P300的通过揭示p53的磷酸化残基与p53的特异性相互作用， Taz 2的近端精氨酸残基，我们能够解释这一结构基础。 重要的信号通路。目前，我们正在追求的复杂的结构， p300 Taz 2结构域与TAD 2，p53的第二个反式激活结构域。这是特别 感兴趣，因为，与第一个不同，相互作用不被磷酸化的蛋白质改变。 p53序列中类似的丝氨酸和苏氨酸残基。最后，我们至少有两个新的 我们正在努力追求的方向。一个是评估一个假定的 稳定C-末端调节结构域中的α-螺旋，并确定 对p53四聚体的稳定性进行修饰。另一种是直接监控在 细胞p53位点特异性化学修饰的动力学及其产物系列 不同类型的细胞应激后分子间的相互作用。最近我们 完成了p300-Taz 2/p53-TAD 2复合物的NMR结构的精细化和解析。

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.