Quantitative analysis of multisite protein-ligand interactions by NMR: binding of intrinsically disordered p53 transactivation subdomains with the TAZ2 domain of CBP.
Quantitative analysis of multisite protein-ligand interactions by NMR: binding of intrinsically disordered p53 transactivation subdomains with the TAZ2 domain of CBP.
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DOI：
10.1021/ja209936u
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发表时间：
2012-02-29
影响因子：
15
通讯作者：
Wright, Peter E.
中科院分区：
文献类型：
--
作者：
Arai, Munehito;Ferreon, Josephine C.;Wright, Peter E.
确定蛋白质-配体相互作用中的亲和力和结合位点对于理解生物系统中的分子机制是必不可少的。在这里，我们结合联合收割机奇异值分解，和全球分析的NMR化学位移扰动所造成的蛋白质-蛋白质相互作用，以确定蛋白质表面上的结合位点的数量和位置，并测量结合亲和力。使用这种方法，我们表明，分离的AD 1和AD 2结合基序，来自内在的无序的N-末端反式激活结构域的肿瘤抑制基因p53，都与TAZ 2结构域的转录辅激活因子CBP在两个结合位点。滴定曲线和线形的模拟表明，只要一级和二级结合过程耦合，核磁滴定法可以准确地测定1~10 nM的一级解离常数。出乎意料的是，TAZ 2疏水表面上的AD 2结合位点与AD 1的结合位点重叠，但AD 2更紧密地结合TAZ 2。这些结果突出了内在无序蛋白质与其靶标之间相互作用的复杂性。此外，AD 2与TAZ 2的结合速率估计为1.7 × 1010 M−1 s−1，接近扩散控制极限，表明内在无序加上互补静电可以显著加速蛋白质结合相互作用。
Determination of affinities and binding sites involved in protein-ligand 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-protein 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 lineshapes show that a primary dissociation constant as small as 1~10 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 × 1010 M−1 s−1, approaching the diffusion-controlled limit and indicating that intrinsic disorder plus complementary electrostatics can significantly accelerate protein binding interactions.
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影响因子：
16.8
作者：
Korennykh, AV;Piccirilli, JA;Correll, CC
通讯作者：
Correll, CC
DOI：
10.1073/pnas.2534828100
发表时间：
2004-01-13
影响因子：
11.1
作者：
Levy, Y;Wolynes, PG;Onuchic, JN
通讯作者：
Onuchic, JN
影响因子：
2.9
作者：
KERN, D;KERN, G;DRAKENBERG, T
通讯作者：
DRAKENBERG, T
影响因子：
2.7
作者：
DELAGLIO, F;GRZESIEK, S;BAX, A
通讯作者：
BAX, A
DOI：
10.1073/pnas.1013078107
发表时间：
2010-11-09
影响因子：
11.1
作者：
Lee, Chul Won;Ferreon, Josephine C.;Wright, Peter E.
通讯作者：
Wright, Peter E.