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

报告人：王泽焕2012.06.20

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。

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。

What is the OMP?

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。

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。

What is the OMP?

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

Synthesize as pre-proteins with an N-terminal signal sequence

An OMP is born!

Ricci DP, Silhavy TJ. Biochim Biophys Acta 2011.

only unfolded proteins can be accommodated by the Sec pathway

The Sec pathway is responsible for transporting the bulk of all exported proteins in E. coli

a targeting element that routes proteins to specific protein translocases embedded in the IM

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.

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.

Fig. 1 Schematic representation of the E. coli Sec transport system

新生肽链

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

ATP 水解推动跨膜运动

信号识别粒子（ SRP ）N-terminal signal

+ 伴侣分子 SecB

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.

Chaperones: always watching, judging

In E.coli , there are parallel pathways of chaperone activityThe 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.

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.

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 Neisseria meningitidis, where the LptD homolog isdispensable 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.

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.

The σE stress response is mediated by a regulated proteolytic cascade triggered by misfolded OMPs.

When misfolded OMPs attack . . .

RseA

RseBActivated 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

an IM-spanning anti-sigma factor that sequesters cytoplasmic σE

Protects RseA from the protease DegS

Be activated

Cleaning up the mess of misfolded OMP aggregates

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? ...