Liu Lu, Mushero Nicole, Hedstrom Lizbeth, Gershenson Anne
Department of Chemistry, Brandeis University, 415 South Street, Waltham, Massachusetts 02454, USA.
Biochemistry. 2006 Sep 12;45(36):10865-72. doi: 10.1021/bi0609568.
Serpins regulate serine proteases by forming metastable covalent complexes with their targets. The protease docks with the serpin and cleaves the serpin's reactive center loop (RCL) forming an acylenzyme intermediate. Cleavage triggers insertion of the RCL into beta sheet A, translocating the attached protease approximately 70 A and disrupting the protease active site, trapping the acylenzyme intermediate. Using single-pair Förster resonance energy transfer (spFRET), we have measured the conformational distributions of trypsin and alpha(1)-proteinase inhibitor (alpha(1)PI) covalent complexes. Bovine trypsin (BTryp) complexes display a single set of conformations consistent with the full translocation of BTryp (E(full)I*). However, the range of spFRET efficiencies is large, suggesting that the region around the trypsin label is mobile. Most complexes between alpha(1)PI variants and the more stable rat trypsin (RTryp) also show a single set of conformations, but the conformational distribution is narrower, indicating less disruption of RTryp. Surprisingly, RTryp complexes containing alpha(1)PI labeled at Cys232 with a cationic fluorophore display two equally populated conformations, E(full)I* and a conformation in which RTryp is only partially translocated (E(part)I*). Destabilizing the RTryp active site, by substituting Ala for Ile16, increases the E(full)I* population. Thus, interactions between anionic RTryp and cationic dyes likely exert a restraining force on alpha(1)PI, increasing the energy needed to translocate trypsin, and this force can be counteracted by active site destabilization. These results highlight the role of protease stability in determining the conformational distributions of protease-serpin covalent complexes and show that full translocation is not required for the formation of metastable complexes.
丝氨酸蛋白酶抑制剂（Serpins）通过与其靶标形成亚稳态共价复合物来调节丝氨酸蛋白酶。蛋白酶与丝氨酸蛋白酶抑制剂对接并切割丝氨酸蛋白酶抑制剂的反应中心环（RCL），形成酰基酶中间体。切割触发RCL插入β折叠A，使附着的蛋白酶移位约70埃并破坏蛋白酶活性位点，捕获酰基酶中间体。使用单对荧光共振能量转移（spFRET），我们测量了胰蛋白酶和α1-抗胰蛋白酶（α1PI）共价复合物的构象分布。牛胰蛋白酶（BTryp）复合物显示出与BTryp完全移位（E（完全）I*）一致的单一组构象。然而，spFRET效率范围很大，表明胰蛋白酶标记周围的区域是可移动的。α1PI变体与更稳定的大鼠胰蛋白酶（RTryp）之间的大多数复合物也显示出单一组构象，但构象分布更窄，表明RTryp的破坏较小。令人惊讶的是，含有在Cys232处用阳离子荧光团标记的α1PI的RTryp复合物显示出两种等量的构象，E（完全）I和RTryp仅部分移位的构象（E（部分）I）。通过将Ala替代Ile16使RTryp活性位点不稳定，增加了E（完全）I*群体。因此，阴离子RTryp与阳离子染料之间的相互作用可能对α1PI施加约束力，增加使胰蛋白酶移位所需的能量，并且这种力可以通过活性位点不稳定来抵消。这些结果突出了蛋白酶稳定性在确定蛋白酶-丝氨酸蛋白酶抑制剂共价复合物构象分布中的作用，并表明形成亚稳态复合物不需要完全移位。
