Campbell Sean T, Carlson Kevin J, Buchholz Carl J, Helmers Mark R, Ghosh Indraneel
Department of Chemistry and Biochemistry, University of Arizona, 1306 East University Boulevard, Tucson, Arizona 85721, United States.
Biochemistry. 2015 Apr 28;54(16):2632-43. doi: 10.1021/bi501505y. Epub 2015 Apr 13.
The recognition of helical BH3 domains by Bcl-2 homology (BH) receptors plays a central role in apoptosis. The residues that determine specificity or promiscuity in this interactome are difficult to predict from structural and computational data. Using a cell free split-luciferase system, we have generated a 276 pairwise interaction map for 12 alanine mutations at the binding interface for three receptors, Bcl-xL, Bcl-2, and Mcl-1, and interrogated them against BH3 helices derived from Bad, Bak, Bid, Bik, Bim, Bmf, Hrk, and Puma. This panel, in conjunction with previous structural and functional studies, starts to provide a more comprehensive portrait of this interactome, explains promiscuity, and uncovers surprising details; for example, the Bcl-xL R139A mutation disrupts binding to all helices but the Bad-BH3 peptide, and Mcl-1 binding is particularly perturbed by only four mutations of the 12 tested (V220A, N260A, R263A, and F319A), while Bcl-xL and Bcl-2 have a more diverse set of important residues depending on the bound helix.
Bcl-2同源（BH）受体对螺旋状BH3结构域的识别在细胞凋亡中起着核心作用。从结构和计算数据中很难预测在这个相互作用组中决定特异性或混杂性的残基。利用无细胞分裂荧光素酶系统，我们针对三种受体Bcl-xL、Bcl-2和Mcl-1的结合界面处的12个丙氨酸突变生成了一个276对相互作用图谱，并用来自Bad、Bak、Bid、Bik、Bim、Bmf、Hrk和Puma的BH3螺旋对它们进行了研究。该研究小组结合之前的结构和功能研究，开始提供这个相互作用组更全面的描述，解释混杂性，并揭示出惊人的细节；例如，Bcl-xL的R139A突变破坏了与所有螺旋的结合，但不影响与Bad-BH3肽的结合，并且在12个测试突变中，只有4个突变（V220A、N260A、R263A和F319A）对Mcl-1的结合有特别大的干扰，而Bcl-xL和Bcl-2根据结合的螺旋不同，有一组更为多样的重要残基。
