How do ATP-independent chaperones assist OMP folding and assembly? Insights from mass spectrometry and other approaches

不依赖 ATP 的分子伴侣如何协助 OMP 折叠和组装？

• 批准号: BB/P000037/1
• 负责人: Sheena Radford
• 金额: $ 58.26万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FP000037%2F1
• 关键词: How; do; ATP; independent; chaperones

Every living cell is surrounded by an envelope, called a membrane, which acts as a barrier to the external environment. These membranes are comprised of lipids and proteins that regulate the entry and exit of molecules in to and out of the cell: in particular regulating the entry of nutrients that ensure that the organism is able to grow and survive. Some bacteria, called gram-negative bacteria, are surrounded by two membranes, one inside the other, called the inner and outer membranes, with a cellular compartment in-between called the periplasm. The outer membrane (OM) of such bacteria contains a distinct selection of lipids, and is crowded with OM proteins (OMPs) that are vital for bacterial survival. New OMPs are made by the bacteria all the time, but the machinery that is used to make these proteins is located in the main compartment of the cell (the cytoplasm), inside the inner membrane. Once made, the OMPs have to undertake a long journey to where they are needed in the cell, the OM. Cellular machinery has evolved to assist this process; in particular, chaperone proteins in the periplasm called Skp and SurA bind to the newly made OMPs and escort them to the OM. The OMPs are then inserted into the OM and folded to the correct structure by a molecular machine called BAM (beta-barrel assembly machinery). Perturbing this pathway leads to a loss of bacterial viability, and therefore represents a potential new avenue for controlling gram-negative pathogens responsible for infections.The structures of the chaperone proteins Skp and SurA, and all the component proteins of BAM, are known. What remains undetermined is how these proteins all work together to make new, folded and functional OMPs. We propose to address this fundamental question by exploiting recent exciting developments in mass spectrometry (MS) (along with other biophysical techniques). Our work will focus on how the two chaperones Skp and SurA bind and recognise their substrate OMPs. It is known that Skp contains a large cavity important for trapping OMPs; however, how this cavity is able to accommodate OMPs of different sizes remains unknown. How SurA, a key chaperone, binds and delivers OMPs for folding into the OM is also unknown in molecular detail. Here we will use MS methods to understand how these chaperones function. We will also use similar approaches to determine the structural organisation of BAM for the first time. Finally, in challenging but exciting experiments, we will investigate how chaperone-bound OMPs are delivered to BAM, and how the subunits of BAM rearrange so that OMPs can be inserted into the OM. Finally, we aim to understand how BAM function can be inhibited. The insights into this essential cellular machinery gained from this work may lead to the identification of much-needed new targets for antibiotics against gram-negative bacteria that cause disease to humans, plants and animals.

每个活细胞都被一层被称为膜的包膜所包围，这层包膜对外部环境起着屏障的作用。这些膜由脂质和蛋白质组成，它们调节分子进出细胞：特别是调节营养物质的进入，确保生物体能够生长和生存。一些被称为革兰氏阴性菌的细菌被两层膜包围，一层膜在另一层膜内，称为内膜和外膜，中间有一个称为周质的细胞隔室。这种细菌的外膜（OM）含有一种独特的脂质选择，并挤满了对细菌生存至关重要的OM蛋白（OMPs）。细菌一直在制造新的omp，但用于制造这些蛋白质的机制位于细胞的主要隔室（细胞质），在内膜内。一旦生成，omp必须经过漫长的旅程才能到达细胞中需要它们的地方，即OM。细胞机制已经进化到协助这一过程；特别是，外周质中称为Skp和SurA的伴侣蛋白与新生成的omp结合并护送它们到OM。然后将omp插入到OM中，并通过称为BAM （β -桶组装机器）的分子机器折叠成正确的结构。干扰这一途径会导致细菌活力的丧失，因此代表了控制导致感染的革兰氏阴性病原体的潜在新途径。伴侣蛋白Skp和SurA的结构以及BAM的所有组成蛋白都是已知的。目前尚不清楚的是，这些蛋白质如何共同作用，产生新的、折叠的、功能性的omp。我们建议利用质谱（MS）（以及其他生物物理技术）的最新令人兴奋的发展来解决这个基本问题。我们的工作将集中在两种伴侣蛋白Skp和SurA如何结合并识别它们的底物omp。已知Skp包含一个对捕获omp很重要的大空腔；然而，这个空腔如何能够容纳不同大小的omp仍然未知。作为关键伴侣的SurA如何结合并递送omp折叠成OM的分子细节也是未知的。在这里，我们将使用质谱方法来了解这些伴侣是如何起作用的。我们还将首次使用类似的方法来确定BAM的结构组织。最后，在具有挑战性但令人兴奋的实验中，我们将研究伴侣结合的omp如何传递到BAM，以及BAM的亚基如何重新排列以使omp可以插入到OM中。最后，我们的目标是了解如何抑制BAM功能。从这项工作中获得的对这种基本细胞机制的见解可能会导致鉴定出急需的针对革兰氏阴性细菌的抗生素的新靶点，革兰氏阴性细菌会导致人类，植物和动物的疾病。

项目成果

Distortion of the bilayer and dynamics of the BAM complex in lipid nanodiscs.

• DOI: 10.1038/s42003-020-01419-w
• 发表时间: 2020-12-14
• 期刊: Communications biology
• 影响因子: 5.9
• 作者: Iadanza MG;Schiffrin B;White P;Watson MA;Horne JE;Higgins AJ;Calabrese AN;Brockwell DJ;Tuma R;Kalli AC;Radford SE;Ranson NA
• 通讯作者: Ranson NA

Interrogating Membrane Protein Structure and Lipid Interactions by Native Mass Spectrometry.

通过天然质谱分析膜蛋白结构和脂质相互作用。

• DOI: 10.1007/978-1-0716-0724-4_11
• 发表时间: 2020
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Hammerschmid D

Rapid Mapping of Protein Interactions Using Tag-Transfer Photocrosslinkers

使用标签转移光交联剂快速绘制蛋白质相互作用图

• DOI: 10.1002/ange.201809149
• 发表时间: 2018
• 期刊: Angewandte Chemie
• 作者: Horne J

Rapid Mapping of Protein Interactions Using Tag-Transfer Photocrosslinkers.

• DOI: 10.1002/anie.201809149
• 发表时间: 2018-12-17
• 期刊: Angewandte Chemie (International ed. in English)
• 作者: Horne JE;Walko M;Calabrese AN;Levenstein MA;Brockwell DJ;Kapur N;Wilson AJ;Radford SE
• 通讯作者: Radford SE