Genetic dissection of the outer membrane secretin PulD: Are there distinct domains for multimerization and secretion specificity?
Genetic dissection of the outer membrane secretin PulD: Are there distinct domains for multimerization and secretion specificity?
复制标题
DOI：
10.1128/jb.181.23.7212-7220.1999
复制
发表时间：
1999-12-01
影响因子：
3.2
通讯作者：
Pugsley, AP
中科院分区：
文献类型：
--
作者：
Guilvout, I;Hardie, KR;Pugsley, AP
使用接头和缺失诱变以及基因融合来探测产酸克雷伯氏菌普鲁兰酶分泌途径十二聚体外膜促胰液素 PulD 的可能结构域结构。在多肽 C 端半部（β 结构域）中靠近或强烈预测和高度保守的两亲性 β 链插入 24 个氨基酸，消除了该蛋白特有的十二烷基硫酸钠 (SDS) 抗性多聚体形成，而其他地方的插入通常对多聚体形成影响较小。然而，单独的 β 结构域不会形成 SDS 抗性多聚体，除非蛋白质的部分 N 末端区域（N 结构域）是反式产生的。除了靠近蛋白质 C 末端的一个插入之外，所有插入都废除了功能。单独的 N 结构域高度不稳定，即使当 β 结构域以反式存在时，也不会形成 SDS 抗性多聚体。我们得出的结论是，β 结构域是多聚体稳定性的主要决定因素，而 N 结构域有助于多聚体形成。 PulD的整个或部分N结构域可以被来自菊欧文氏菌果胶酸裂合酶分泌途径的OutD促胰液素的相应区域取代，而不消除支链淀粉酶的分泌。这表明 PulD 的 N 结构域不参与底物识别，这与 OutD 的 N 结构域所提出的作用相反，OutD 与 E. chrysanthemi 分泌的果胶酸裂合酶特异性结合（V. E. Shevchik、J. Robert-Badouy 和 G. Condemine, EMBO J. 16:3007-3016, 1997）。
Linker and deletion mutagenesis and gene fusions were used to probe the possible domain structure of the dodecameric outer membrane secretin PulD from the pullulanase secretion pathway of Klebsiella oxytoca. Insertions of 24 amino acids close to or within strongly predicted and highly conserved amphipathic beta strands in the C-terminal half of the polypeptide (the beta domain) abolished sodium dodecyl sulfate (SDS)-resistant multimer formation that is characteristic of this protein, whereas insertions elsewhere generally had less dramatic effects on multimer formation. However, the beta domain alone did not form SDS-resistant multimers unless part of the N-terminal region of the protein (the N domain) was produced in trans. All of the insertions except one, close to the C terminus of the protein, abolished function. The N domain alone was highly unstable and did not form SDS-resistant multimers even when the beta domain was present in trans. We conclude that the beta domain is a major determinant of multimer stability and that the N domain contributes to multimer formation. The entire or part of the N domain of PulD could be replaced by the corresponding region of the OutD secretin from the pectate lyase secretion pathway of Erwinia chrysanthemi without abolishing pullulanase secretion. This suggests that the N domain of PulD is not involved in substrate recognition, contrary to the role proposed for the N domain of OutD, which binds specifically to pectate lyase secreted by E. chrysanthemi (V. E. Shevchik, J. Robert-Badouy, and G. Condemine, EMBO J. 16:3007-3016, 1997).