Trimeric structure of major outer membrane proteins homologous to OmpA in Porphyromonas gingivalis
Trimeric structure of major outer membrane proteins homologous to OmpA in Porphyromonas gingivalis
复制标题
DOI：
10.1128/jb.187.3.902-911.2005
复制
发表时间：
2005-02-01
影响因子：
3.2
通讯作者：
Yoshimura, F
中科院分区：
文献类型：
--
作者：
Nagano, K;Read, EK;Yoshimura, F
牙龈卟啉单胞菌ATCC 33277的主要外膜蛋白Pgm 6（41 kDa）和Pgm 7（40 kDa）分别由开放阅读框pg 0695和pg 0694编码，其形成单个操纵子。Pgm 6和Pgm 7（Pgm 6/7）在C-末端区域与大肠杆菌OmpA具有高度相似性，并且预测在N-末端区域形成八链β-桶。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，Pgm 6/7出现为条带，表观分子量为40和120 kDa，有和没有还原剂，分别表明单体和三聚体。为了验证预测的三聚体结构和功能的Pgm 6/7，我们构建了三个突变体与pg 0695，pg 0694，或两者缺失。双突变体不产生Pgm 6/7。单缺失突变体似乎含有较少的Pgm 7和Pgm 6，并形成同源三聚体，迁移稍快（115 kDa）和较慢（130 kDa），分别比野生型Pgm 6/7在非还原条件下。部分消化的Pgm 6/7的N-末端氨基酸测序和质谱分析仅检测到来自Pgm 6和Pgm 7的片段。有或没有还原剂的二维，对角线电泳和化学交联实验清楚地表明，Pgm 6/7主要通过分子间二硫键形成稳定的异源三聚体。此外，生长迟缓和逮捕的三个突变体和增加其外膜的通透性表明，Pgm 6/7发挥重要作用的外膜的完整性。基于脂质体溶胀实验的结果，这些蛋白质可能作为细胞壁的稳定剂而不是作为该生物体中的主要孔蛋白。
The major outer membrane proteins Pgm6 (41 kDa) and Pgm7 (40 kDa) of Porphyromonas gingivalis ATCC 33277 are encoded by open reading frames pg0695 and pg0694, respectively, which form a single operon. Pgm6 and Pgm7 (Pgm6/7) have a high degree of similarity to Escherichia coli OmpA in the C-terminal region and are predicted to form eight-stranded beta-barrels in the N-terminal region. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Pgm6/7 appear as bands with apparent molecular masses of 40 and 120 kDa, with and without a reducing agent, suggesting a monomer and trimer, respectively. To verify the predicted trimeric structure and function of Pgm6/7, we constructed three mutants with pg0695, pg0694, or both deleted. The double mutant produced no Pgm6/7. The single-deletion mutants appeared to contain less Pgm7 and Pgm6 and to form homotrimers that migrated slightly faster (115 kDa) and slower (130 kDa), respectively, than wild-type Pgm6/7 under nonreducing conditions. N-terminal amino acid sequencing and mass spectrometry analysis of partially digested Pgm6/7 detected only fragments from Pgm6 and Pgm7. Two-dimensional, diagonal electrophoresis and chemical cross-linking experiments with or without a reducing agent clearly showed that Pgm6/7 mainly form stable heterotrimers via intermolecular disulfide bonds. Furthermore, growth retardation and arrest of the three mutants and increased permeability of their outer membranes indicated that Pgm6/7 play an important role in outer membrane integrity. Based on results of liposome swelling experiments, these proteins are likely to function as a stabilizer of the cell wall rather than as a major porin in this organism.