Making a beta-barrel: assembly of outer membrane proteins in Gram-negative bacteria

1. Making a beta-barrel: assembly of outer membrane proteins in Gram-negative bacteria 报告人：王泽焕 2012.06.20

2. What is the OMP? 细菌表面蛋白与外膜蛋白的区别 ? 革兰氏阴性菌与革兰阳性菌相比较其细胞壁有一种特殊成分叫外膜。外膜由内到外由脂蛋白、脂质双层和脂多糖三部分组成，脂质双层内镶嵌的蛋白质称为外膜蛋白。 某些革兰氏阳性菌表面有一些特殊的表面蛋白；如金黄色葡萄球菌的A蛋白，A链球菌的M蛋白等。

3. What is the OMP? 定义：外膜蛋白（OMP）是革兰氏阴性菌特有的，存在于外 膜之中及与外膜有关的所有蛋白的总称。

4. What is the OMP? 外膜蛋白的结构 外膜蛋白不是由跨生物膜的α螺旋组成，而是由反平行的β桶结构组成，不同的外膜蛋白的β-桶由不同偶数个β-折叠片组成，从8个到22个不等。

5. What is the OMP? 外膜蛋白的功能 外膜蛋白能使革兰氏阴性菌抵抗恶劣环境，与此 同时，镶嵌蛋白大部分与细菌细胞的关键功能起到作用。

6. An OMP is born! The OMP assembly pathway in E. coli . Outer membrane proteins (OMPs) are synthesized as unfolded precursors in the cytoplasm and translocated across the inner membrane (IM) via the Sec translocase. Once in the periplasm, chaperones recognize unfolded OMPs and prevent them from forming misfolded aggregates. The multi-subunit Bam complex folds and inserts OMPs into the outer membrane

7. An OMP is born! Synthesize as pre-proteins with an N-terminal signal sequence a targeting element that routes proteins to specific protein translocases embedded in the IM The Sec pathway is responsible for transporting the bulk of all exported proteins in E. coli only unfolded proteins can be accommodated by the Sec pathway Ricci DP, Silhavy TJ. Biochim Biophys Acta 2011.

8. The Sec pathway Sec translocase（Sec 转运酶） Sec转运酶是一个多组分的蛋白质复合体，膜蛋白三聚体 SecYEG 及水解ATP的动力蛋白 SecA 构成 Sec 转运酶的核心。整合膜蛋白 SecD，SecF 和 YajC 形成了一个复合体亚单位，可与 SecYEG 相连并稳定SecA蛋白的膜结合形式。 SecYEG——转运通道 SecA——亲水蛋白质，具有ATP 酶活性，是Sec蛋白质转运途径中的“动力泵” SecG——小分子的具有柔性膜蛋白 SecB——疏水的表面和新合成的多肽结合 Anastassios E. Federation of European Biochemical Societies , 2000, 476 : 18 − 21. Sugai R, Takemae K, Tokuda H. J Biol Chem , 2007, 282 (40), 29540 − 29548.

9. 信号识别粒子（SRP） N-terminal signal 新生肽链 + 伴侣分子 SecB SRP与它的膜受体 FtsY 结合 SecB 与膜上的SecA结合 成熟肽链直接被SecA识别 起始蛋白转运过程 成熟肽链由SecB转移至SecA SecYEG 组成通道进行转运 Fig. 1 Schematic representation of the E. coli Sec transport system Note: some preproteins are delivered to SecA via SecB, while others directly interact with SecA without the participation of SecB. Upon interacting with the inner membranebound SecYEG, SecA may or may not dissociate to monomer. ATP水解 推动跨膜运动

10. Chaperones: always watching, judging Since beta-barrel proteins will ultimately reside in the OM, it is not surprising that these proteins are prone to aggregation in aqueous environments. To prevent aggregation and misfolding these hydrophobic residues must be shielded from the aqueous periplasm. In E.coli , there are parallel pathways of chaperone activity The chaperone SurA functions in one pathway and the chaperone Skp and the chaperone DegP in the other. Merdanovic M, Clausen T, Kaiser M. Annu Rev Microbiol 2011, 65:149-168.

11. Chaperones: always watching, judging SurA: SurA can function to assemble all OMPs efficiently, and some OMPs, including the essential LPS assembly factor LptD prefer this pathway. Skp: no OMPs that prefer the Skp pathway have been identified. However, in the absence of SurA, Skp can assemble most OMPs efficiently.

12. Chaperones: always watching, judging The importance of SurA is evidenced by the increased permeability, OMP assembly defects, and envelope stress factor induction observed in surA mutants. In Neisseriameningitidis, where the LptD homolog is dispensable for growth, the OMP assembly defects observed in a surA mutant are mild. Such defects are much more pronounced in a skp mutant. Suggesting that Skp might be the major periplasmic chaperone in this organism. Sklar JG, Wu T, Kahne D, Silhavy TJ. Genes Dev 2007, 21:2473-2484 Volokhina EB, Grijpstra J, Stork M, Tommassen J. Bos MPJ Bacteriol 2011, 193 :1612-1621.

13. When misfolded OMPs attack . . . The journey an unfolded OMP takes from the IM to the OM is filled with peril. It is well established that the accumulation of misfolded protein aggregates serves as an inducing cue for cellular stress responses including the σE and Cpx systems.

14. When misfolded OMPs attack . . . The σE stress response is mediated by a regulated proteolytic cascade triggered by misfolded OMPs. an IM-spanning anti-sigma factor that sequesters cytoplasmic σE RseA Protects RseA from the protease DegS Be activated Cleaning up the mess of misfolded OMP aggregates RseB Activated by binding DegS to specific C-terminal residues of misfolded OMPs Beta-strand motif (BSM) also in the C-terminal portion of misfolded OMPs by binding RseB

15. What’s next? The last decade has seen an incredible increase in our understanding of OMP biogenesis. However, despite these advances, the mechanism by which beta-barrels are folded and inserted into the OM bilayer still lacks detail. Is the barrel domain folded before membrane insertion? Are different classes of OMPs assembled in the same way? ...