National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences , 15 Datun Road, Beijing 100101, China.
Biochemistry. 2013 Sep 17;52(37):6324-34. doi: 10.1021/bi400690m. Epub 2013 Sep 4.
One of the transcription-independent mechanisms of the tumor suppressor p53 discovered in recent years involves physical interaction between p53 and proteins of the Bcl-2 family. In this paper, significant differences between the interaction of p53 with Mcl-1 and Bcl-xL were demonstrated by NMR spectroscopy and isothermal titration calorimetry. Bcl-xL was found to bind strongly to the p53 DNA-binding domain (DBD) with a dissociation constant (Kd) of ~600 nM, whereas Mcl-1 binds to the p53 DBD weakly with a dissociation constant in the mM range. In contrast, the p53 transactivation domain (TAD) binds weakly to Bcl-xL with a Kd ~ 300-500 μM and strongly to Mcl-1 with a Kd ~ 10-20 μM. NMR titrations indicate that although the p53 TAD binds to the BH3-binding grooves of both Bcl-xL and Mcl-1, Bcl-xL prefers to bind to the first subdomain (TAD1) in the p53 TAD, and Mcl-1 prefers to bind to the second subdomain (TAD2). Therefore, Mcl-1 and Bcl-xL have different p53-binding profiles. This indicates that the detailed interaction mechanisms are different, although both Mcl-1 and Bcl-xL can mediate transcription-independent cytosolic roles of p53. The revealed differences in binding sites and binding affinities should be considered when BH3 mimetics are used in cancer therapy development.
近年来发现的肿瘤抑制因子 p53 的转录非依赖性机制之一涉及 p53 与 Bcl-2 家族蛋白之间的物理相互作用。在本文中，通过 NMR 光谱和等温滴定量热法证明了 p53 与 Mcl-1 和 Bcl-xL 的相互作用存在显著差异。Bcl-xL 与 p53 DNA 结合域（DBD）强烈结合，解离常数（Kd）约为 600 nM，而 Mcl-1 与 p53 DBD 弱结合，解离常数在 mM 范围内。相比之下，p53 转录激活域（TAD）与 Bcl-xL 弱结合，Kd 约为 300-500 μM，与 Mcl-1 强结合，Kd 约为 10-20 μM。NMR 滴定表明，尽管 p53 TAD 与 Bcl-xL 和 Mcl-1 的 BH3 结合槽结合，但 Bcl-xL 更倾向于与 p53 TAD 的第一个亚域（TAD1）结合，而 Mcl-1 更倾向于与第二个亚域（TAD2）结合。因此，Mcl-1 和 Bcl-xL 具有不同的 p53 结合谱。这表明，尽管 Mcl-1 和 Bcl-xL 都可以介导 p53 的转录非依赖性细胞质作用，但详细的相互作用机制是不同的。在开发用于癌症治疗的 BH3 模拟物时，应考虑结合位点和结合亲和力的差异。
