Bochkareva Elena, Kaustov Lilia, Ayed Ayeda, Yi Gwan-Su, Lu Ying, Pineda-Lucena Antonio, Liao Jack C C, Okorokov Andrei L, Milner Jo, Arrowsmith Cheryl H, Bochkarev Alexey
Banting and Best Department of Medical Research & Department of Medical Genetics and Microbiology, University of Toronto, 112 College Street, Toronto, Ontario, Canada M5G 1L6.
Proc Natl Acad Sci U S A. 2005 Oct 25;102(43):15412-7. doi: 10.1073/pnas.0504614102. Epub 2005 Oct 17.
One of many protein-protein interactions modulated upon DNA damage is that of the single-stranded DNA-binding protein, replication protein A (RPA), with the p53 tumor suppressor. Here we report the crystal structure of RPA residues 1-120 (RPA70N) bound to the N-terminal transactivation domain of p53 (residues 37-57; p53N) and, by using NMR spectroscopy, characterize two mechanisms by which the RPA/p53 interaction can be modulated. RPA70N forms an oligonucleotide/oligosaccharide-binding fold, similar to that previously observed for the ssDNA-binding domains of RPA. In contrast, the N-terminal p53 transactivation domain is largely disordered in solution, but residues 37-57 fold into two amphipathic helices, H1 and H2, upon binding with RPA70N. The H2 helix of p53 structurally mimics the binding of ssDNA to the oligonucleotide/oligosaccharide-binding fold. NMR experiments confirmed that both ssDNA and an acidic peptide mimicking a phosphorylated form of RPA32N can independently compete the acidic p53N out of the binding site. Taken together, our data suggest a mechanism for DNA damage signaling that can explain a threshold response to DNA damage.
在DNA损伤时被调节的众多蛋白质-蛋白质相互作用之一，是单链DNA结合蛋白复制蛋白A（RPA）与p53肿瘤抑制因子之间的相互作用。在此我们报道了与p53的N端反式激活结构域（第37-57位氨基酸残基；p53N）结合的RPA第1-120位氨基酸残基（RPA70N）的晶体结构，并通过核磁共振光谱法，描述了两种可调节RPA/p53相互作用的机制。RPA70N形成了一个寡核苷酸/寡糖结合折叠结构，类似于先前在RPA的单链DNA结合结构域中观察到的结构。相比之下，p53的N端反式激活结构域在溶液中大多无序，但在与RPA70N结合时，第37-57位氨基酸残基折叠成两个两亲性螺旋，即H1和H2。p53的H2螺旋在结构上模拟了单链DNA与寡核苷酸/寡糖结合折叠结构的结合。核磁共振实验证实，单链DNA和模拟RPA32N磷酸化形式的酸性肽都能独立地将酸性的p53N从结合位点上竞争下来。综合来看，我们的数据提示了一种DNA损伤信号传导机制，该机制可以解释对DNA损伤的阈值反应。
