Huang Yihua, Rich Rebecca L, Myszka David G, Wu Hao
Department of Biochemistry, Weill Medical College and Graduate School of Medical Sciences of Cornell University, New York, NY 10021, USA.
J Biol Chem. 2003 Dec 5;278(49):49517-22. doi: 10.1074/jbc.M310061200. Epub 2003 Sep 25.
The inhibitor of apoptosis proteins (IAP) are endogenous caspase inhibitors in the metazoan and characterized by the presence of baculoviral IAP repeats (BIR). X-linked IAP (XIAP) contains three BIR domains and directly inhibits effector caspases such as caspase-7 via a linker_BIR2 fragment and initiator caspases such as caspase-9 via the BIR3 domain. A mitochondrial protein Smac/DIABLO, which is released during apoptosis, antagonizes XIAP-mediated caspase inhibition by interacting directly with XIAP. Here, using glutathione S-transferase pulldown and caspase activity assay, we show that Smac is ineffective in relieving either caspase-7 or caspase-9 inhibition by XIAP domain fragments. In addition, Smac forms a ternary complex with caspase-7 and linker_BIR2, suggesting that Smac/linker_BIR2 interaction does not sterically exclude linker_BIR2/caspase-7 interaction. However, Smac is effective in removing caspase-7 and caspase-9 inhibition by XIAP fragments containing both the BIR2 and BIR3 domains. Surface plasmon resonance measurements show that Smac interacts with the BIR2 or BIR3 domain in micromolar dissociation constants. On the other hand, Smac interacts with an XIAP construct containing both BIR2 and BIR3 domains in a subnanomolar dissociation constant by the simultaneous interaction of the Smac dimer with the BIR2 and BIR3 domains of a single XIAP molecule. This 2:1 Smac/XIAP interaction not only possesses enhanced affinity but also sterically excludes XIAP/caspase-7 interaction, demonstrating the requirement of both BIR2 and BIR3 domains for Smac to relieve XIAP-mediated caspase inhibition.
凋亡抑制蛋白（IAP）是后生动物体内的内源性半胱天冬酶抑制剂，其特征是存在杆状病毒IAP重复序列（BIR）。X连锁IAP（XIAP）包含三个BIR结构域，通过连接子_BIR2片段直接抑制效应半胱天冬酶如半胱天冬酶-7，并通过BIR3结构域抑制起始半胱天冬酶如半胱天冬酶-9。一种在凋亡过程中释放的线粒体蛋白Smac/DIABLO，通过与XIAP直接相互作用来拮抗XIAP介导的半胱天冬酶抑制作用。在此，我们利用谷胱甘肽S-转移酶下拉实验和半胱天冬酶活性测定表明，Smac在缓解XIAP结构域片段对半胱天冬酶-7或半胱天冬酶-9的抑制方面无效。此外，Smac与半胱天冬酶-7和连接子_BIR2形成三元复合物，这表明Smac/连接子_BIR2相互作用在空间上并不排除连接子_BIR2/半胱天冬酶-7相互作用。然而，Smac在去除含有BIR2和BIR3结构域的XIAP片段对半胱天冬酶-7和半胱天冬酶-9的抑制方面是有效的。表面等离子体共振测量表明，Smac以微摩尔解离常数与BIR2或BIR3结构域相互作用。另一方面，Smac通过Smac二聚体与单个XIAP分子的BIR2和BIR3结构域同时相互作用，以亚纳摩尔解离常数与含有BIR2和BIR3结构域的XIAP构建体相互作用。这种2:1的Smac/XIAP相互作用不仅具有增强的亲和力，而且在空间上排除了XIAP/半胱天冬酶-7相互作用，证明了Smac缓解XIAP介导的半胱天冬酶抑制需要BIR2和BIR3结构域。
