Baek Je-Hyun, Im Hana, Kang Un-Beom, Seong Ki Moon, Lee Cheolju, Kim Joon, Yu Myeong-Hee
Functional Proteomics Center, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, Korea.
Protein Sci. 2007 Sep;16(9):1842-50. doi: 10.1110/ps.072911607. Epub 2007 Jul 27.
The native form of serpins (serine protease inhibitors) is a metastable conformation, which converts into a more stable form upon complex formation with a target protease. It has been suggested that movement of helix-F (hF) and the following loop connecting to strand 3 of beta-sheet A (thFs3A) is critical for such conformational change. Despite many speculations inferred from analysis of the serpin structure itself, direct experimental evidence for the mobilization of hF/thFs3A during the inhibition process is lacking. To probe the mechanistic role of hF and thFs3A during protease inhibition, a disulfide bond was engineered in alpha(1)-antitrypsin, which would lock the displacement of thFs3A from beta-sheet A. We measured the inhibitory activity of each disulfide-locked mutant and its heat stability against loop-sheet polymerization. Presence of a disulfide between thFs3A and s5A but not between thFs3A and s3A caused loss of the inhibitory activity, suggesting that displacement of hF/thFs3A from strand 5A but not from strand 3A is required during the inhibition process. While showing little influence on the inhibitory activity, the disulfide between thFs3A and s3A retarded loop-sheet polymerization significantly. This successful protein engineering of alpha(1)-antitrypsin is expected to be of value in clinical applications. Based on our current studies, we propose that the reactive-site loop of a serpin glides through between s5A and thFs3A for the full insertion into beta-sheet A while a substantial portion of the interactions between hF and s3A is kept intact.
丝氨酸蛋白酶抑制剂（serpins）的天然形式是一种亚稳态构象，在与靶蛋白酶形成复合物后会转变为更稳定的形式。有人提出，F螺旋（hF）及其后连接β折叠A的3链的环（thFs3A）的移动对于这种构象变化至关重要。尽管从丝氨酸蛋白酶抑制剂结构本身的分析中推断出了许多推测，但在抑制过程中hF/thFs3A移动的直接实验证据仍然缺乏。为了探究hF和thFs3A在蛋白酶抑制过程中的作用机制，在α1-抗胰蛋白酶中设计了一个二硫键，该二硫键将锁定thFs3A从β折叠A的位移。我们测量了每个二硫键锁定突变体的抑制活性及其对环片聚合的热稳定性。thFs3A和s5A之间存在二硫键而thFs3A和s3A之间不存在二硫键会导致抑制活性丧失，这表明在抑制过程中需要hF/thFs3A从5A链而非3A链位移。虽然对抑制活性影响不大，但thFs3A和s与3A之间的二硫键显著延迟了环片聚合。α1-抗胰蛋白酶的这种成功的蛋白质工程有望在临床应用中具有价值。基于我们目前的研究，我们提出丝氨酸蛋白酶抑制剂的反应位点环在s5A和thFs3A之间滑动以完全插入β折叠A，而hF和s3A之间的大部分相互作用保持完整。
