Understanding the ABCs of multi drug resistance - tying the knot on the antibacterial peptide ABC transporter McjD

了解多重耐药性的ABC - 抗菌肽ABC转运蛋白McjD喜结连理

• 批准号: MR/N020103/1
• 负责人: Konstantinos Beis
• 金额: $ 64.07万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FN020103%2F1
• 关键词: Understanding; ABCs; multi; drug; resistance

In recent years, outbreaks of E. coli infections in UK and Europe highlight the importance of understanding the disease mechanisms of such important pathogens and biomolecules. Bacteria have developed mechanisms to confer resistance to current antibiotics and one of the routes of resistance are membrane proteins that export drugs from the cell, called exporters. Membrane proteins represent around 30% of the proteomes of most organisms and more than 40% of drug targets and yet few structures of these molecules have been solved by X-ray crystallography. These proteins are usually embedded in oil like environment making them very difficult to work with. We first need to isolate them from the membrane using lipid mimics such as detergents. Membrane proteins usually have many functions and bacteria have developed mechanisms to utilise them in order to extrude antibiotics through these proteins. In order to develop new treatments it is important to understand the molecular mechanism of these important biological molecules. Using X-ray crystallography, we can gain a detailed understanding of the mechanism of these proteins; we need to grow crystals and expose them to X-rays in order to obtain the molecular structure. Certain drugs in the market are based on peptide chemistry and the bacteria have evolved these transporters to provide them with immunity by extruding them from inside the cell. One such family of proteins are the ATP-binding cassette transporters (ABC transporters) that are powered by the hydrolysis of ATP. Our group has recently solved the X-ray structure of an ABC exporter that is involved in antibacterial transport, McjD, which confers resistance to the antibacterial peptide MccJ25. We know that when the antibacterial peptide MccJ25 and ATP bind to McjD, the ABC exporter can adopt different conformations (a movement that mimics opening/closing of a gate) in order to extrude the toxic compound. In our crystal structure, McjD adopts a new conformation, nucleotide-bound outward occluded; the portion of the protein that sits in the membrane has a closed cavity where the MccJ25 can fit in. The structural information and biochemical experiments of this membrane protein will provide an important insight on the binding and recognition of antibacterial peptides and allow us to get a detailed picture of its mechanism. We aim to trap the transporter in different conformations and characterise it in the presence of the antibacterial peptides in order to reveal the mode of binding and transport. The biochemical experiments will try identifying (1) the binding and recognition of the peptide by McjD and (2) the conformational changes that McjD undergoes during peptide binding and transport (before ligand binding, occluded state and after the hydrolysis of ATP (resetting the system)). Understanding the structure and function of these important biological molecules will contribute to the understanding of the relationship between membrane protein and substrate recognition and provide valuable information to structural biology and pharmacology.

近年来，我国爆发了E.英国和欧洲的大肠杆菌感染突出了了解这些重要病原体和生物分子的疾病机制的重要性。细菌已经发展出了对当前抗生素产生耐药性的机制，耐药性的途径之一是从细胞中输出药物的膜蛋白，称为输出者。膜蛋白占大多数生物体蛋白质组的30%左右，占药物靶点的40%以上，但这些分子的结构很少被X射线晶体学解决。这些蛋白质通常嵌入在油样环境中，使它们非常难以处理。我们首先需要使用脂质模拟物如去污剂将它们从膜上分离出来。膜蛋白通常具有许多功能，细菌已经开发出利用它们的机制，以便通过这些蛋白质挤出抗生素。为了开发新的治疗方法，重要的是要了解这些重要的生物分子的分子机制。使用X射线晶体学，我们可以详细了解这些蛋白质的机制;我们需要生长晶体并将其暴露于X射线以获得分子结构。市场上的某些药物是基于肽化学的，细菌已经进化出这些转运蛋白，通过将它们从细胞内挤出来为它们提供免疫力。一个这样的蛋白质家族是ATP结合盒转运蛋白（ABC转运蛋白），其由ATP水解提供动力。我们的团队最近解决了ABC出口商的X射线结构，该出口商参与抗菌转运，McjD，其赋予抗菌肽MccJ 25的抗性。我们知道，当抗菌肽MccJ 25和ATP与McjD结合时，ABC输出器可以采用不同的构象（模拟门的打开/关闭的运动）以挤出有毒化合物。在我们的晶体结构中，McjD采用了一种新的构象，核苷酸结合向外封闭;位于膜中的蛋白质部分具有一个封闭的空腔，MccJ 25可以容纳其中。该膜蛋白的结构信息和生物化学实验将为抗菌肽的结合和识别提供重要的见解，并使我们能够详细了解其机制。我们的目标是捕获不同构象的转运蛋白，并在抗菌肽的存在下对其进行修饰，以揭示结合和转运的模式。生化实验将尝试鉴定（1）McjD对肽的结合和识别，以及（2）McjD在肽结合和转运过程中（配体结合前、封闭状态和ATP水解后（重置系统））经历的构象变化。了解这些重要生物分子的结构和功能将有助于了解膜蛋白与底物识别的关系，并为结构生物学和药理学提供有价值的信息。

项目成果

Widespread emergence of OmpK36 loop 3 insertions among multidrug-resistant clones of Klebsiella pneumoniae

肺炎克雷伯菌多重耐药克隆中广泛出现 OmpK36 环 3 插入

• DOI: 10.1101/2022.02.07.479342
• 发表时间: 2022
• 作者: David S

PELDOR/DEER: An Electron Paramagnetic Resonance Method to Study Membrane Proteins in Lipid Bilayers.

PELDOR/DEER：一种研究脂质双层膜蛋白的电子顺磁共振方法。

• DOI: 10.1007/978-1-0716-0724-4_15
• 发表时间: 2020
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Peter MF

Structural basis for antibacterial peptide self-immunity by the bacterial ABC transporter McjD.

• DOI: 10.15252/embj.201797278
• 发表时间: 2017-10-16
• 期刊: The EMBO journal
• 作者: Bountra K;Hagelueken G;Choudhury HG;Corradi V;El Omari K;Wagner A;Mathavan I;Zirah S;Yuan Wahlgren W;Tieleman DP;Schiemann O;Rebuffat S;Beis K
• 通讯作者: Beis K

Widespread emergence of OmpK36 loop 3 insertions among multidrug-resistant clones of Klebsiella pneumoniae.

• DOI: 10.1371/journal.ppat.1010334
• 发表时间: 2022-07
• 期刊: PLoS pathogens
• 影响因子: 6.7

Experimental phasing with vanadium and application to nucleotide-binding membrane proteins.

• DOI: 10.1107/s2052252520012312
• 发表时间: 2020-11-01
• 期刊: IUCrJ
• 影响因子: 3.9
• 作者: El Omari K;Mohamad N;Bountra K;Duman R;Romano M;Schlegel K;Kwong HS;Mykhaylyk V;Olesen C;Moller JV;Bublitz M;Beis K;Wagner A
• 通讯作者: Wagner A