Charbonneau Marie-Ève, Janvore Julie, Mourez Michael
From the Canada Research Chair on Bacterial Animal Diseases, Université de Montréal, Faculté de Médecine Vétérinaire, 3200 Sicotte, St.-Hyacinthe, Québec J2S 7C6, Canada.
From the Canada Research Chair on Bacterial Animal Diseases, Université de Montréal, Faculté de Médecine Vétérinaire, 3200 Sicotte, St.-Hyacinthe, Québec J2S 7C6, Canada.
J Biol Chem. 2009 Jun 19;284(25):17340-17351. doi: 10.1074/jbc.M109.010108. Epub 2009 Apr 27.
The cleavage of the autotransporter adhesin involved in diffuse adherence (AIDA-I) of Escherichia coli yields a membrane-embedded fragment, AIDAc, and an extracellular fragment, the mature AIDA-I adhesin. The latter remains noncovalently associated with AIDAc but can be released by heat treatment. In this study we determined the mechanism of AIDA-I cleavage. We showed that AIDA-I processing is an autocatalytic event by monitoring the in vitro cleavage of an uncleaved mutant protein isolated from inclusion bodies. Furthermore, by following changes in circular dichroism spectra and protease resistance of the renaturated protein, we showed that the cleavage of the protein is correlated with folding. With site-directed deletions, we showed that the catalytic activity of the protein lies in a region encompassing amino acids between Ala-667 and Thr-953, which includes the conserved junction domain of some autotransporters. With site-directed point mutations, we also found that Asp-878 and Glu-897 are involved in the processing of AIDA-I and that a mutation preserving the acidic side chain of Asp-878 was tolerated, giving evidence that this carboxylic acid group is directly involved in catalysis. Last, we confirmed that cleavage of AIDA-I is intramolecular. Our results unveil a new mechanism of auto-processing in the autotransporter family.
参与大肠杆菌弥漫性黏附的自转运黏附素（AIDA-I）的裂解产生一个膜嵌入片段AIDAc和一个细胞外片段，即成熟的AIDA-I黏附素。后者与AIDAc保持非共价结合，但可通过热处理释放。在本研究中，我们确定了AIDA-I裂解的机制。通过监测从包涵体中分离出的未裂解突变蛋白的体外裂解，我们表明AIDA-I的加工是一个自催化事件。此外，通过跟踪复性蛋白的圆二色光谱变化和蛋白酶抗性，我们表明该蛋白的裂解与折叠相关。通过定点缺失，我们表明该蛋白的催化活性位于一个包含Ala-667至Thr-953之间氨基酸的区域，其中包括一些自转运蛋白的保守连接结构域。通过定点突变，我们还发现Asp-878和Glu-897参与AIDA-I的加工，并且保留Asp-878酸性侧链的突变是可耐受的，这表明该羧酸基团直接参与催化。最后，我们证实AIDA-I的裂解是分子内的。我们的结果揭示了自转运蛋白家族中一种新的自我加工机制。
