Haberz Peter, Arai Munehito, Martinez-Yamout Maria A, Dyson H Jane, Wright Peter E
Department of Integrative Structural and Computational Biology and The Skaggs Institute of Chemical Biology, The Scripps Research Institute, La Jolla, California.
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro, Tokyo, 153-8902, Japan.
Protein Sci. 2016 Dec;25(12):2256-2267. doi: 10.1002/pro.3059. Epub 2016 Oct 15.
Many viruses deregulate the cell and force transcription of viral genes by competing with cellular proteins for binding to the transcriptional co-activators CREB-binding protein (CBP) and p300. Through its interactions with CBP/p300 and the retinoblastoma protein, the adenovirus (AdV) early region 1A (E1A) oncoprotein hijacks the cell cycle and, in rodents, transforms the cell; the mechanistic and structural basis for these effects remain unclear. In this study we compare the affinity of protein constructs from the E1A proteins from two adenovirus serotypes, non-oncogenic AdV5 and highly oncogenic AdV12, for binding to the nuclear receptor coactivator binding domain (NCBD) of CBP. NMR spectra show that the E1A constructs from both serotypes are intrinsically disordered in the free state and that each contains three homologous binding sites for the NCBD, one in the N-terminal region and two within conserved region 1 (CR1) of E1A. The binding sites in CR1 correspond to the motifs that bind the retinoblastoma protein and the TAZ2 domain of CBP/p300. The E1A and NCBD peptides fold synergistically upon complex formation. Binding affinities determined from NMR titrations show that, although the overall affinities for AdV5 and AdV12 E1A are comparable, there are significant differences between the two E1A serotypes in the relative strength with which their constituent interaction motifs bind to the NCBD. The individual E1A interaction motifs were unable to compete effectively with p53 for binding to the NCBD and both the N-terminal region and CR1 region of E1A are required for efficient competition with p53.
许多病毒通过与细胞蛋白竞争结合转录共激活因子CREB结合蛋白（CBP）和p300来破坏细胞调控并促使病毒基因转录。腺病毒（AdV）早期区域1A（E1A）癌蛋白通过与CBP/p300和视网膜母细胞瘤蛋白相互作用，劫持细胞周期，并在啮齿动物中使细胞发生转化；这些效应的机制和结构基础仍不清楚。在本研究中，我们比较了来自两种腺病毒血清型（非致癌性AdV5和高致癌性AdV12）的E1A蛋白构建体与CBP的核受体共激活因子结合结构域（NCBD）的结合亲和力。核磁共振光谱表明，两种血清型的E1A构建体在游离状态下都是内在无序的，并且每种构建体都包含三个与NCBD同源的结合位点，一个在N端区域，两个在E1A的保守区域1（CR1）内。CR1中的结合位点对应于与视网膜母细胞瘤蛋白以及CBP/p300的TAZ2结构域结合的基序。E1A和NCBD肽在形成复合物时协同折叠。通过核磁共振滴定确定的结合亲和力表明，尽管AdV5和AdV12 E1A的总体亲和力相当，但两种E1A血清型在其组成相互作用基序与NCBD结合的相对强度上存在显著差异。单个E1A相互作用基序无法有效地与p53竞争结合NCBD，E1A的N端区域和CR1区域对于与p53的有效竞争都是必需的。
