Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu, 9, Madrid, 28040, Spain.
J Mol Recognit. 2012 Dec;25(12):665-73. doi: 10.1002/jmr.2240.
According to biochemical assays, the Bcl-2 protein Diva from mouse regulates programmed cell death by heterodimerizing with other members of the family and by interacting with the apoptotic protease-activating factor Apaf-1. In typical Bcl-2 heterodimers, peptide fragments comprising the Bcl-2 homology domain 3 (BH3 domain) of proapoptotic members are capable of forming functional complexes with prosurvival proteins. High-resolution structural studies have revealed that the BH3 peptide forms an α-helix positioned in a canonical hydrophobic cleft of the antiapoptotic protein. Because Diva shows mutations in conserved residues within this area, it has been proposed to have a different interacting surface. However, we showed previously that Diva binds through the canonical groove the BH3 peptide of the human Bcl-2 killing member Harakiri. To further test Diva's binding capabilities, here we show Nuclear Magnetic Resonance (NMR) data, indicating that Diva binds peptides derived from the BH3 domain of several other proapoptotic Bcl-2 proteins, including mouse Harakiri, Bid, Bak and Bmf. We have measured the binding affinities of the heterodimers, which show significant variability. Structural models of the protein-peptide complexes based on NMR chemical shift perturbation data indicate that the binding surface is analogous. These models do not rely on NMR NOE (Nuclear Overhauser Effect) data, and thus our results can only suggest that the complexes share similar intermolecular interactions. However, the observed affinity differences correlate with the α-helical population of the BH3-peptides obtained from circular dichroism experiments, which highlights a role of conformational selection in the binding mechanism. Altogether, our results shed light on important factors governing Diva-BH3 peptide molecular recognition mode.
根据生化分析，来自老鼠的 Bcl-2 蛋白 Diva 通过与家族的其他成员形成异二聚体以及与凋亡蛋白酶激活因子 Apaf-1 相互作用来调节程序性细胞死亡。在典型的 Bcl-2 异二聚体中，包含促凋亡成员的 Bcl-2 同源结构域 3（BH3 结构域）的肽片段能够与生存蛋白形成功能复合物。高分辨率结构研究表明，BH3 肽形成一个 α-螺旋，位于抗凋亡蛋白的典型疏水性裂缝中。由于 Diva 在该区域的保守残基中显示出突变，因此有人提出它具有不同的相互作用表面。然而，我们之前曾表明，Diva 通过经典沟槽与人类 Bcl-2 杀伤成员 Harakiri 的 BH3 肽结合。为了进一步测试 Diva 的结合能力，我们在这里展示了核磁共振（NMR）数据，表明 Diva 通过经典沟槽与几种其他促凋亡 Bcl-2 蛋白的 BH3 结构域衍生的肽结合，包括鼠 Harakiri、Bid、Bak 和 Bmf。我们已经测量了异二聚体的结合亲和力，其具有显著的可变性。基于 NMR 化学位移扰动数据的蛋白质-肽复合物的结构模型表明，结合表面是类似的。这些模型不依赖于 NMR NOE（核 Overhauser 效应）数据，因此我们的结果只能表明复合物具有相似的分子间相互作用。然而，观察到的亲和力差异与从圆二色性实验获得的 BH3-肽的α-螺旋群体相关，这突出了构象选择在结合机制中的作用。总的来说，我们的结果阐明了决定 Diva-BH3 肽分子识别模式的重要因素。
