Split luciferase complementary assay has been used to investigate the effect of WD domain deletion on Apaf-1 oligomerization. Apaf-1 is an adaptor molecule in formation of apoptosome that activates caspase-9, an activation that is a key event in the mitochondrial cell death pathway. Structural studies suggest that normally Apaf-1 is held in an inactive conformation by intramolecular interactions between Apaf-1’s nucleotide binding domain and one of its WD40 domains (WD1). In the prevailing model of Apaf-1 activation, cytochrome c binds to sites in WD1 and in Apaf-1’s second WD40 domain (WD2), moving WD1 and WD2 closer together and rotating WD1 away from the nucleotide binding domain. This allows Apaf-1 to bind dATP or ATP and to form the apoptosome, which activates caspase-9. This model predicts that cytochrome c binding to both WD domains is necessary for apoptosome formation and that an Apaf-1 with only WD1 will be locked in an inactive conformation that cannot be activated by cytochrome c. Here we investigated the effect of removing one WD domain (Apaf-1 1–921) on Apaf-1 interactions and caspase activation. Apaf-1 1–921 could not activate caspase-9, even in the presence of cytochrome c. These data show that a single WD domain is sufficient to lock Apaf-1 in an inactive state and this state cannot be altered by cytochrome c.

分裂荧光素酶互补测定已用于研究WD域缺失对Apaf-1寡聚的影响。Apaf-1是凋亡小体形成的衔接子分子，可激活caspase-9，该激活是线粒体细胞死亡途径中的关键事件。结构研究表明，正常情况下，Apaf-1通过Apaf-1核苷酸结合结构域与其WD40结构域之一（WD1）之间的分子内相互作用而保持非活性构象。在Apaf-1激活的流行模型中，细胞色素c与WD1和Apaf-1的第二个WD40域（WD2）中的位点结合，使WD1和WD2靠得更近，并使WD1远离核苷酸结合域。这允许Apaf-1结合dATP或ATP并形成凋亡小体，从而激活caspase-9。该模型预测，细胞色素c与两个WD域的结合对于凋亡小体形成是必要的，并且只有WD1的Apaf-1将被锁定在无法被细胞色素c激活的非活性构象中。在这里，我们研究了删除一个WD域（Apaf-1 1–921）对Apaf-1相互作用和caspase激活的影响。即使存在细胞色素c，Apaf-1 1–921也不能激活caspase-9。这些数据表明，单个WD结构域足以将Apaf-1锁定在非活动状态，并且该状态不能被细胞色素c改变。