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，我们关注的是通过将MDM2和MDMX蛋白结合到N端的反式激活结构域来使p53失活。这项工作导致了两种类型的竞争性抑制分子的发展。首先，基于聚N-取代甘氨酸支架，证明了这种类肽可以针对蛋白质靶标进行设计。第二种更容易制造的分子是基于一种新型的N-酰基多胺(NPA)支架。后一种分子经过优化，具有与众所周知的MDM2抑制剂Nutlin(Hoffman-La Roche)相当的结合亲和力。然而，我们的抑制剂优于Nutlin，对MDM2和MDMX都有效。随后，我们集中研究了P53与组蛋白乙酰转移酶共激活物CREB结合蛋白(CBP)和p300的功能相互作用。染色质结合的p53将这些蛋白质招募到基因启动子上，导致组蛋白的局部乙酰化，从而解开转录所需的染色质。CBP和P300各自由七个不同的域组成，排列在一个共同的体系结构中。其中有两个转录适配子锌结合结构域，Taz1(C/H1)和Taz2(C/H3)，它们介导对转录至关重要的蛋白质-蛋白质相互作用。虽然已知这两个结构域都与P53的两个反式激活结构域(TAD1和TAD2)相互作用，但结构细节尚不清楚。在冯汉桥和白亚文博士(LBMB/NCI)的合作下，我们首次阐明了P53的TAD1与P300的Taz2结构域的相互作用结构。在该复合体中，P53肽形成一个短螺旋，并通过延伸的表面与Taz2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体中观察到的不同，最明显的是与MDM2和MDMX的复合体。虽然络合物主要由疏水键稳定，但静电相互作用也起到了作用。我们进一步的研究涉及核磁共振、突变和热力学，表明复合体的结构是如何改变的，并在Ser15和Thr18残基的p53磷酸化后进一步稳定，这是已知的CBP和p300招募的翻译后修饰信号。通过揭示P53的磷酸化残基与Taz2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。目前，我们正在寻找p300Taz2结构域与P53的第二个反式激活结构域TAD2的复合体的结构。这一点特别令人感兴趣，因为与第一种不同，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要紧锣密鼓地追求。一种是评估在C末端调节域中可能形成稳定的α-螺旋，并确定修饰对P53四聚体稳定的影响。另一种是直接在细胞内监测P53特定部位化学修饰的动力学，以及在不同类型的细胞压力下产生的一系列分子相互作用。最近，我们完成了p300-Taz2/P53-TAD2复合体的核磁共振结构的提纯和分析。

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.