Elliott P R, Abrahams J P, Lomas D A
Department of Medicine, University of Cambridge, UK.
J Mol Biol. 1998 Jan 23;275(3):419-25. doi: 10.1006/jmbi.1997.1458.
alpha 1-Antitrypsin is the archetypal member of the serine proteinase inhibitor or serpin superfamily. Members of the family show structural homology based on a dominant A beta-sheet and a mobile reactive centre loop. Our recent crystal structure of alpha 1-antitrypsin stabilized with a point mutation showed the loop to be in a canonical inhibitory conformation in the absence of significant insertion into the A beta-sheet. It could be argued that the stabilizing mutation may induce the reactive centre loop to adopt an artificial, and unrepresentative, conformation and the finding seems to be at variance with studies assessing rates of peptide insertion into the A beta-sheet and limited proteolysis of the reactive loop. Here we present a 2.9 A structure of recombinant wild-type alpha 1-antitrypsin with no stabilizing mutations. Again, the reactive loop is in a canonical conformation in the absence of significant insertion into the A beta-sheet. A stabilizing salt bridge between P5 glutamate and arginine residues 196, 223 and 281, already identified in the mutant, provides strong evidence that this conformation is not an artefact of crystallization but represents the conformation of the circulating inhibitor in vivo. Comparison with the structure of alpha 1-antitrypsin stabilized with the Phe51Leu mutation indicates that the increased thermal stability of the mutant results from enhanced packing of aromatic residues in the hydrophobic core of the molecule. The structure of wild-type alpha 1-antitrypsin reveals a hydrophobic pocket between s2A and helices D and E that is filled on reactive loop insertion and the formation of biologically relevant loop-sheet polymers. This pocket may provide a target for rational drug design to prevent the formation of polymers and the associated plasma deficiency, liver cirrhosis and emphysema.
α1-抗胰蛋白酶是丝氨酸蛋白酶抑制剂（serpin）超家族的典型成员。该家族成员基于一个占主导地位的Aβ-折叠和一个可移动的反应中心环表现出结构同源性。我们最近通过点突变稳定化的α1-抗胰蛋白酶晶体结构显示，在没有显著插入Aβ-折叠的情况下，该环处于典型的抑制构象。可以认为，稳定化突变可能诱导反应中心环采用一种人为的、不具代表性的构象，而且这一发现似乎与评估肽插入Aβ-折叠的速率以及反应环的有限蛋白水解的研究结果不一致。在此，我们展示了无稳定化突变的重组野生型α1-抗胰蛋白酶的2.9埃结构。同样，在没有显著插入Aβ-折叠的情况下，反应环处于典型构象。在突变体中已鉴定出的P5谷氨酸与精氨酸残基196、223和281之间的一个稳定盐桥，有力地证明了这种构象不是结晶假象，而是代表了体内循环抑制剂的构象。与用Phe51Leu突变稳定化的α1-抗胰蛋白酶结构的比较表明，突变体热稳定性的提高源于分子疏水核心中芳香族残基堆积的增强。野生型α1-抗胰蛋白酶的结构揭示了在s2A与螺旋D和E之间有一个疏水口袋，在反应环插入以及形成生物学相关的环-片聚合物时该口袋会被填满。这个口袋可能为合理药物设计提供一个靶点，以防止聚合物的形成以及相关的血浆缺乏、肝硬化和肺气肿。
