Pazgier Marzena, Liu Min, Zou Guozhang, Yuan Weirong, Li Changqing, Li Chong, Li Jing, Monbo Juahdi, Zella Davide, Tarasov Sergey G, Lu Wuyuan
Institute of Human Virology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA.
Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4665-70. doi: 10.1073/pnas.0900947106. Epub 2009 Mar 2.
The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53--a cellular process initiated by MDM2 and/or MDMX binding to the N-terminal transactivation domain of p53. MDM2 and MDMX in many tumors confer p53 inactivation and tumor survival, and are important molecular targets for anticancer therapy. We screened a duodecimal peptide phage library against site-specifically biotinylated p53-binding domains of human MDM2 and MDMX chemically synthesized via native chemical ligation, and identified several peptide inhibitors of the p53-MDM2/MDMX interactions. The most potent inhibitor (TSFAEYWNLLSP), termed PMI, bound to MDM2 and MDMX at low nanomolar affinities--approximately 2 orders of magnitude stronger than the wild-type p53 peptide of the same length (ETFSDLWKLLPE). We solved the crystal structures of synthetic MDM2 and MDMX, both in complex with PMI, at 1.6 A resolution. Comparative structural analysis identified an extensive, tightened intramolecular H-bonding network in bound PMI that contributed to its conformational stability, thus enhanced binding to the 2 oncogenic proteins. Importantly, the C-terminal residue Pro of PMI induced formation of a hydrophobic cleft in MDMX previously unseen in the structures of p53-bound MDM2 or MDMX. Our findings deciphered the structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX, shedding new light on structure-based rational design of different classes of p53 activators for potential therapeutic use.
癌蛋白MDM2和MDMX对肿瘤抑制蛋白p53的活性和稳定性起负调控作用，这一细胞过程由MDM2和/或MDMX与p53的N端反式激活结构域结合启动。在许多肿瘤中，MDM2和MDMX会导致p53失活并使肿瘤存活，它们是抗癌治疗的重要分子靶点。我们针对通过天然化学连接法化学合成的人MDM2和MDMX的位点特异性生物素化p53结合结构域筛选了一个十二肽噬菌体文库，并鉴定出几种p53-MDM2/MDMX相互作用的肽抑制剂。最有效的抑制剂（TSFAEYWNLLSP），称为PMI，以低纳摩尔亲和力与MDM2和MDMX结合，比相同长度的野生型p53肽（ETFSDLWKLLPE）强约2个数量级。我们以1.6埃的分辨率解析了与PMI形成复合物的合成MDM2和MDMX的晶体结构。比较结构分析确定了结合态PMI中广泛且紧密的分子内氢键网络，这有助于其构象稳定性，从而增强了与这两种致癌蛋白的结合。重要的是，PMI的C端残基Pro在MDMX中诱导形成了一个疏水裂缝，这在与p53结合的MDM2或MDMX结构中未曾见过。我们的研究结果揭示了高亲和力肽抑制p53与MDM2和MDMX相互作用的结构基础，为基于结构的不同类型p53激活剂的合理设计提供了新线索，有望用于治疗。
