Faculté des Sciences et Techniques, INRA, UMR1061 Unité de génétique Moléculaire Animale, Université de Limoges, FR 3503 GEIST, 87060 Limoges, France.
Protein Sci. 2012 Jul;21(7):977-86. doi: 10.1002/pro.2078. Epub 2012 May 18.
The family of serpins is known to fold into a metastable state that is required for the proteinase inhibition mechanism. One of the consequences of this conformational flexibility is the tendency of some mutated serpins to form polymers, which occur through the insertion of the reactive center loop of one serpin molecule into the A-sheet of another. This "A-sheet polymerization" has remained an attractive explanation for the molecular mechanism of serpinopathies. Polymerization of serpins can also take place in vitro under certain conditions (e.g., pH or temperature). Surprisingly, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, bovSERPINA3-3 extracted from skeletal muscle or expressed in Escherichia coli was mainly observed as a homodimer. Here, in this report, by site-directed mutagenesis of recombinant bovSERPINA3-3, with substitution D371A, we demonstrate the importance of D371 for the intermolecular linkage observed in denaturing and reducing conditions. This residue influences the electrophoretic and conformational properties of bovSERPINA3-3. By structural modeling of mature bovSERPINA3-3, we propose a new "non-A-sheet swap" model of serpin homodimer in which D371 is involved at the molecular interface.
丝氨酸蛋白酶抑制剂家族（serpins）已知会折叠成一种亚稳态，这种状态是蛋白酶抑制机制所必需的。这种构象灵活性的后果之一是，一些突变丝氨酸蛋白酶倾向于形成聚合物，这是通过将一个丝氨酸蛋白酶分子的反应中心环插入另一个丝氨酸蛋白酶的 A 片层来实现的。这种“A 片层聚合”仍然是丝氨酸蛋白酶病分子机制的一个有吸引力的解释。在某些条件下（例如 pH 值或温度），丝氨酸蛋白酶也可以在体外聚合。令人惊讶的是，在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳上，从骨骼肌中提取或在大肠杆菌中表达的 bovSERPINA3-3 主要观察到作为同源二聚体。在这里，在本报告中，通过对重组 bovSERPINA3-3 的定点突变，用 D371A 取代，我们证明了 D371 对变性和还原条件下观察到的分子间连接的重要性。该残基影响 bovSERPINA3-3 的电泳和构象特性。通过对成熟 bovSERPINA3-3 的结构建模，我们提出了一个新的丝氨酸蛋白酶同源二聚体“非 A 片层交换”模型，其中 D371 参与分子界面。
