Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158, USA.
Proc Natl Acad Sci U S A. 2013 May 21;110(21):8477-82. doi: 10.1073/pnas.1306759110. Epub 2013 May 6.
Procaspase-3 (P3) and procaspase-7 (P7) are activated through proteolytic maturation to form caspase-3 (C3) and caspase-7 (C7), respectively, which serve overlapping but nonredundant roles as the executioners of apoptosis in humans. However, it is unclear if differences in P3 and P7 maturation mechanisms underlie their unique biological functions, as the structure of P3 remains unknown. Here, we report structures of P3 in a catalytically inactive conformation, structures of P3 and P7 bound to covalent peptide inhibitors that reveal the active conformation of the zymogens, and the structure of a partially matured C7:P7 heterodimer. Along with a biochemical analysis, we show that P3 is catalytically inactive and matures through a symmetric all-or-nothing process. In contrast, P7 contains latent catalytic activity and matures through an asymmetric and tiered mechanism, suggesting a lower threshold for activation. Finally, we use our structures to design a selection strategy for conformation specific antibody fragments that stimulate procaspase activity, showing that executioner procaspase conformational equilibrium can be rationally modulated. Our studies provide a structural framework that may help to explain the unique roles of these important proapoptotic enzymes, and suggest general strategies for the discovery of proenzyme activators.
前胱冬蛋白酶-3（P3）和前胱冬蛋白酶-7（P7）通过蛋白水解成熟分别激活为胱冬蛋白酶-3（C3）和胱冬蛋白酶-7（C7），它们在人类中作为凋亡的执行者发挥重叠但非冗余的作用。然而，尚不清楚 P3 和 P7 成熟机制的差异是否是其独特生物学功能的基础，因为 P3 的结构仍然未知。在这里，我们报告了 P3 在无催化活性构象下的结构，P3 和 P7 与共价肽抑制剂结合的结构，揭示了酶原的活性构象，以及部分成熟的 C7：P7 杂二聚体的结构。通过生化分析，我们表明 P3 无催化活性，并且通过对称的全有或全无过程成熟。相比之下，P7 包含潜在的催化活性，并且通过不对称和分层机制成熟，这表明激活的阈值较低。最后，我们使用我们的结构设计了一种针对构象特异性抗体片段的选择策略，该策略可刺激前胱冬蛋白酶活性，表明执行器前胱冬蛋白酶构象平衡可以被合理调节。我们的研究提供了一个结构框架，可能有助于解释这些重要的促凋亡酶的独特作用，并为发现酶原激活剂提供了一般策略。
