H2020Индивидуална стипендия2015–2017

FLUOROKEF · Incorporation of unnatural fluorinated amino acids to probe the function of the bacterial efflux system Kef in a cellular setting.

„Хоризонт 2020“ — Действия „Мария Склодовска-Кюри“

Период: 2015-10-01 → 2017-09-30
Финансиране от ЕС: 183 455 €
Участници: 1
Схема: MSCA-IF-EF-ST

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Накратко на български

Бактериалната система Kef, която изхвърля калий от клетката, се анализира чрез добавяне на флуорни аминокиселини за проследяване на работата ѝ. Разбирането на този механизъм помага при разработването на нови антимикробни средства, тъй като хората нямат подобни протеини.

Този кратък обзор е генериран от изкуствен интелект

Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.

Резултати накратко

Incorporation of unnatural fluorinated amino acids to probe the function of the bacterial efflux system Kef in a cellular setting.

The ligand-gated bacterial potassium (K+) efflux system, Kef, is inhibited by glutathione (GSH) and activated by glutathione-S-conjugates (GS-X). Kef is evolutionarily conserved in many bacterial species and functions to protect bacteria against toxic electrophiles. Its vital role in cell homeostasis and lack of Kef homologs in humans make it a promising target for new antimicrobials. Our previous studies have suggested that the F441 residue of Escherichia coli KefC (EcKefC) plays a crucial role in Kef ligand gating. This mechanism is conserved in many bacterial species and F448 of Shewanella denitrificans Kef (SdKef) serves the same function. Although biochemical, crystallographic and genetic studies have been conducted on Kef, the dynamic aspect of the Kef activation mechanism has never been explored. While conventional 2D nuclear magnetic resonance (NMR) techniques have been useful in probing protein dynamics, the large size of homodimeric Kef proteins presents a formidable challenge. Here, we overcame this hurdle by site-specifically incorporating 19F-containing non-standard amino acids (nsAAs) into a SdKef C-terminal domain construct (SdKefQCTD) and by using protein-observed 19F NMR (PrOF-NMR) spectroscopy to monitor dynamics of the resulting fluorinated protein. This has enabled the dynamic mechanism of Kef activation to be directly observed in both a purified Kef protein and a bacterial cell lysate for the first time. We found that chemically-synthesized Kef activators can dynamically displace the F448 gating residue, which in turn leads to Kef activation, while inhibitory binders of Kef do not. Moreover, this novel fluoro-Kef platform provides new insights into not only binding but also function of Kef ligands. In conjunction with molecular dynamics (MD) simulations, we further validated the dynamic mechanism of Kef activation. This work not only sheds light on the Kef activation mechanism, but also provides an invaluable tool for the future discovery of novel antibacterial compounds that target Kef. Using E. coli kef knockout cell strain genetically modified to express recombinant SdKef as a model, compounds that show binding affinities to Kef were examined for their antimicrobial activity by the Kirby-Bauer disk diffusion method. We found that Kef activator can kill/inhibit the model bacteria. These results might pave the way to develop a new antibiotic in the future.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз

Цел на проекта

The potassium efflux system, Kef, protects bacteria against the detrimental effects of electrophilic compounds via acidification of the cytoplasm. Its vital role in maintaining cell homeostasis makes Kef a promising target for antibiotics, but such drugs can be developed only with a molecular understanding of Kef activation. Our work indicates that phenylalanine residue 441 (F441) in Escherichia coli is crucial for the activation of K+ efflux. This mechanism is conserved, and F448 of Shewanella denitrificans Kef (SdKef) has the same function. This work employed X-ray crystallographic studies using a truncated construct of the E. coli Kef soluble C-terminal domain (EcKefCTD) and biophysical studies conducted on the SdKef C-terminal domain (SdKefCTD). While the crystallographic studies have been essential in hypothesis generation, they lack the dynamic aspect that is required to understand the mechanism of Kef activation. Although the use of a truncated construct has allowed development of biophysical assays, we wish to extend our work to focus on the full-length channel. To achieve these aims we will employ whole cell 19F NMR to probe the function of Kef.To determine if 19F NMR can be used to monitor F448 conformation, we will employ amber stop codon technology to replace F448 of SdKef with 4-trifluromethyl-L-phenylalanine (tfmF). We will monitor the dynamics of F448tfmF by 19F NMR upon binding of activators and inhibitors, using our library of SdKef ligands. To gain an understanding of Kef activation in cells, we will quantitatively determine binding affinities of the Kef ligands to SdKef(F448tfmF), in E. coli transformants using in-cell 19F NMR. This direct determination of binding inside the cells will be more physiologically relevant than the in vitro KD data. To assess the antibiotic activity of compounds that show cellular activity against Kef, we will employ the Kirby-Bauer disc diffusion method to screen for compounds that can kill bacteria.

Оригинален текст от CORDIS (на английски).

Участници

THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD · OxfordКоординаторОбединеното кралство