Molecular basis of protein translocation through outer membrane porins

蛋白质通过外膜孔蛋白易位的分子基础

• 批准号: BB/L021234/1
• 负责人: Colin Kleanthous
• 金额: $ 95万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL021234%2F1
• 关键词: Molecular; basis; protein; translocation; through

Gram-negative bacteria are surrounded by two membranes, the outer most of which (usually referred to as the 'outer membrane') is a highly effective barrier against toxic molecules; for example, bile salts which exist within the gut of a mammal. The effectiveness of the outer membrane is a double-edged sword for the organism since it is also very effective at keeping out essential nutrients such as sugars, which the organism needs to grow and divide. Hence all organisms that have an outer membrane also have specialised proteins within this membrane whose job it is to allow the exchange of nutrients and metabolites with the environment. These proteins are known as porins, which are barrel-like membrane proteins that have a hole or pore running through them which traverses the membrane. There are many barrel proteins in the outer membrane of Gram-negative bacteria (it is estimated that 2-3% of the E. coli genome encode such proteins) which serve a variety of functions. Those that allow the exchange of nutrients are usually referred to as general or classical porins, the best understood of which are the proteins OmpF and OmpC. These porins are also the major routes by which commonly used antibiotics (e.g. ampicillin) diffuse into the cell. Indeed, the channels of these porins are frequently found to be mutated in multidrug resistant bacteria. The focus of this research proposal is a novel function we have recently discovered for the general porin OmpF which shows that porins can do more than just let small molecules diffuse through their pores. We have discovered that OmpF can also be used to allow proteins to pass (translocate) into the cell, as long as the protein is unfolded (i.e. a random coil) and can snake its way through the narrow pores of the porin. Even more remarkably, this translocating protein snakes through two of the three pores of OmpF, which is composed of three barrel subunits; i.e. it goes into the cell then comes back out again. Given the importance of porins to the physiology of bacteria and eukaryotes (mammals and plants have porins in the outer membranes of some of their organelles) our discovery has important ramifications for our understanding of the biology of organisms that have outer membranes since it shows that proteins can exploit their porins. Proteins have functions which are usually imparted by their having a three dimensional structure or fold. Although the proteins that pass through OmpF are unfolded polypeptides and so by implication have no function, these can become functional by virtue of their being able to bind to other molecules such as proteins, and hence in this way alter cellular behaviour. The twin aims of this proposal are to understand the molecular basis for protein translocation through the porin OmpF in the gut bacterium Escherichia coli (determining for example how much polypeptide can pass through the pore) and to discover how common this phenomenon is by looking at other porins and porins from other organisms. Another important question to be investigated is what drives protein translocation through porins? This is an important question since the outer membrane has no energy source to call upon in order to drag a protein into a cell (unlike the inner most membrane which does) and so answering this question will not only provide us with fundamental new insights into bacterial porins themselves but also the outer membrane in which they reside.

革兰氏阴性菌被两层膜包围，其中最外层（通常称为“外膜”）是对抗有毒分子的高效屏障;例如，存在于哺乳动物肠道内的胆汁盐。外膜的有效性对生物体来说是一把双刃剑，因为它也非常有效地阻止了生物体生长和分裂所需的必需营养素，如糖。因此，所有具有外膜的生物体在膜内也有专门的蛋白质，其工作是允许营养物质和代谢物与环境交换。这些蛋白质被称为孔蛋白，它们是具有贯穿膜的孔或孔的桶状膜蛋白。革兰氏阴性菌的外膜中含有大量的桶蛋白（据估计，2-3%的E。大肠杆菌基因组编码这样的蛋白质），其具有多种功能。那些允许营养交换的蛋白质通常被称为一般或经典的孔蛋白，其中最好理解的是蛋白质OmpF和OmpC。这些孔蛋白也是常用抗生素（例如氨苄青霉素）扩散到细胞中的主要途径。事实上，这些孔蛋白的通道经常被发现在多药耐药细菌中发生突变。这项研究计划的重点是我们最近发现的一般孔蛋白OmpF的新功能，这表明孔蛋白可以做的不仅仅是让小分子通过它们的孔扩散。我们已经发现，OmpF也可以用于允许蛋白质通过（易位）进入细胞，只要蛋白质是未折叠的（即无规卷曲），并且可以通过孔蛋白的狭窄孔蜿蜒前进。更值得注意的是，这种转运蛋白会穿过OmpF的三个孔中的两个，OmpF由三个桶亚基组成;也就是说，它进入细胞，然后再出来。考虑到孔蛋白对细菌和真核生物生理学的重要性（哺乳动物和植物的一些细胞器的外膜上有孔蛋白），我们的发现对我们理解具有外膜的生物体的生物学具有重要的影响，因为它表明蛋白质可以利用它们的孔蛋白。蛋白质具有通常由其具有三维结构或折叠赋予的功能。虽然通过OmpF的蛋白质是未折叠的多肽，因此暗示没有功能，但这些蛋白质可以通过与其他分子如蛋白质结合而变得有功能，从而以这种方式改变细胞行为。该提案的双重目的是了解蛋白质通过肠道细菌大肠杆菌中的孔蛋白OmpF移位的分子基础（例如确定有多少多肽可以通过孔），并通过观察其他孔蛋白和来自其他生物体的孔蛋白来发现这种现象有多普遍。另一个需要研究的重要问题是什么驱动蛋白质通过孔蛋白移位？这是一个重要的问题，因为外膜没有能量来源来将蛋白质拖进细胞（不像最内层的膜那样），因此回答这个问题不仅会为我们提供关于细菌孔蛋白本身的基本新见解，也会为我们提供它们所居住的外膜。

项目成果

Correction to "Directional Porin Binding of Intrinsically Disordered Protein Sequences Promotes Colicin Epitope Display in the Bacterial Periplasm".

• DOI: 10.1021/acs.biochem.8b00864
• 发表时间: 2018-09-04
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Housden NG;Rassam P;Lee S;Samsudin F;Kaminska R;Sharp C;Goult JD;Francis ML;Khalid S;Bayley H;Kleanthous C
• 通讯作者: Kleanthous C

Directional Porin Binding of Intrinsically Disordered Protein Sequences Promotes Colicin Epitope Display in the Bacterial Periplasm.

固有无序蛋白序列的定向孔蛋白结合可促进细菌周期中的结肠表位。

• DOI: 10.1021/acs.biochem.8b00621
• 发表时间: 2018-07-24
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Housden NG;Rassam P;Lee S;Samsudin F;Kaminska R;Sharp C;Goult JD;Francis ML;Khalid S;Bayley H;Kleanthous C
• 通讯作者: Kleanthous C

Lipid binding attenuates channel closure of the outer membrane protein OmpF.

• DOI: 10.1073/pnas.1721152115
• 发表时间: 2018-06-26
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Liko I;Degiacomi MT;Lee S;Newport TD;Gault J;Reading E;Hopper JTS;Housden NG;White P;Colledge M;Sula A;Wallace BA;Kleanthous C;Stansfeld PJ;Bayley H;Benesch JLP;Allison TM;Robinson CV
• 通讯作者: Robinson CV

High-resolution mass spectrometry of small molecules bound to membrane proteins.

• DOI: 10.1038/nmeth.3771
• 发表时间: 2016-04
• 期刊: Nature methods
• 影响因子: 48
• 作者: Gault J;Donlan JA;Liko I;Hopper JT;Gupta K;Housden NG;Struwe WB;Marty MT;Mize T;Bechara C;Zhu Y;Wu B;Kleanthous C;Belov M;Damoc E;Makarov A;Robinson CV
• 通讯作者: Robinson CV