Xue Y, Björquist P, Inghardt T, Linschoten M, Musil D, Sjölin L, Deinum J
Department of Inorganic Chemistry, Göteborg University, Sweden.
Structure. 1998 May 15;6(5):627-36. doi: 10.1016/s0969-2126(98)00064-1.
Plasminogen activator inhibitor type 1 (PAI-1) is an important endogenous regulator of the fibrinolytic system. Reduction of PAI-1 activity has been shown to enhance dissolution of blood clots. Like other serpins, PAI-1 binds covalently to a target serine protease, thereby irreversibly inactivating the enzyme. During this process the exposed reactive-centre loop of PAI-1 is believed to undergo a conformational change becoming inserted into beta sheet A of the serpin. Incubation with peptides from the reactive-centre loop transform serpins into a substrate for their target protease. It has been hypothesised that these peptides bind to beta sheet A, thereby hindering the conformational rearrangement leading to loop insertion and formation of the stable serpin-protease complex.
We report here the 1.95 A X-ray crystal structure of a complex of a glycosylated mutant of PAI-1, PAI-1-ala335Glu, with two molecules of the inhibitory reactive-centre loop peptide N-Ac-TVASS-NH2. Both bound peptide molecules are located between beta strands 3A and 5A of the serpin. The binding kinetics of the peptide inhibitor to immobilised PAI-1-Ala335Glu, as monitored by surface plasmon resonance, is consistent with there being two different binding sites.
This is the first reported crystal structure of a complex formed between a serpin and a serpin inhibitor. The localisation of the inhibitory peptide in the complex strongly supports the theory that molecules binding in the space between beta strands 3A and 5A of a serpin are able to prevent insertion of the reactive-centre loop into beta sheet A, thereby abolishing the ability of the serpin to irreversibly inactivate its target enzyme. The characterisation of the two binding sites for the peptide inhibitor provides a solid foundation for computer-aided design of novel, low molecular weight PAI-1 inhibitors.
纤溶酶原激活物抑制剂1（PAI - 1）是纤维蛋白溶解系统的重要内源性调节因子。已表明降低PAI - 1活性可增强血凝块溶解。与其他丝氨酸蛋白酶抑制剂一样，PAI - 1与靶丝氨酸蛋白酶共价结合，从而不可逆地使该酶失活。在此过程中，PAI - 1暴露的反应中心环被认为会发生构象变化，插入丝氨酸蛋白酶抑制剂的β - 折叠A中。与来自反应中心环的肽一起孵育会将丝氨酸蛋白酶抑制剂转化为其靶蛋白酶的底物。据推测，这些肽与β - 折叠A结合，从而阻碍导致环插入和稳定的丝氨酸蛋白酶抑制剂 - 蛋白酶复合物形成的构象重排。
我们在此报告了糖基化突变体PAI - 1 - ala335Glu与两个抑制性反应中心环肽N - Ac - TVASS - NH2分子形成的复合物的1.95 Å X射线晶体结构。两个结合的肽分子都位于丝氨酸蛋白酶抑制剂的β链3A和5A之间。通过表面等离子体共振监测，肽抑制剂与固定化的PAI - 1 - Ala335Glu的结合动力学与存在两个不同结合位点一致。
这是首次报道的丝氨酸蛋白酶抑制剂与丝氨酸蛋白酶抑制剂抑制剂形成的复合物的晶体结构。复合物中抑制性肽的定位有力地支持了这样一种理论，即在丝氨酸蛋白酶抑制剂的β链3A和5A之间的空间中结合的分子能够阻止反应中心环插入β - 折叠A，从而消除丝氨酸蛋白酶抑制剂不可逆地使其靶酶失活的能力。肽抑制剂两个结合位点的表征为新型低分子量PAI - 1抑制剂的计算机辅助设计提供了坚实基础。
