Quantitative Analysis of Multisite Protein鈥揕igand Interactions by NMR: Binding of Intrinsically Disordered p53 Transactivation Subdomains with the TAZ2 Domain of CBP
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- 作者：Munehito Arai ; Josephine C. Ferreon ; Peter E. Wright
- 刊名：The Journal of the American Chemical Society
- 出版年：2012
- 出版时间：February 29, 2012
- 年：2012
- 卷：134
- 期：8
- 页码：3792-3803
- 全文大小：662K
- 年卷期：v.134,no.8(February 29, 2012)
- ISSN：1520-5126
文摘
Determination of affinities and binding sites involved in protein鈥搇igand interactions is essential for understanding molecular mechanisms in biological systems. Here we combine singular value decomposition and global analysis of NMR chemical shift perturbations caused by protein鈥損rotein interactions to determine the number and location of binding sites on the protein surface and to measure the binding affinities. Using this method we show that the isolated AD1 and AD2 binding motifs, derived from the intrinsically disordered N-terminal transactivation domain of the tumor suppressor p53, both interact with the TAZ2 domain of the transcriptional coactivator CBP at two binding sites. Simulations of titration curves and line shapes show that a primary dissociation constant as small as 1鈥?0 nM can be accurately estimated by NMR titration methods, provided that the primary and secondary binding processes are coupled. Unexpectedly, the site of binding of AD2 on the hydrophobic surface of TAZ2 overlaps with the binding site for AD1, but AD2 binds TAZ2 more tightly. The results highlight the complexity of interactions between intrinsically disordered proteins and their targets. Furthermore, the association rate of AD2 to TAZ2 is estimated to be 1.7 脳 1010 M鈥? s鈥?, approaching the diffusion-controlled limit and indicating that intrinsic disorder plus complementary electrostatics can significantly accelerate protein binding interactions.