Periodic Reporting for period 1 - FLUOROKEF (Incorporation of unnatural fluorinated amino acids to probe the function of the bacterial efflux system Kef in a cellular setting.)
Reporting period: 2015-10-01 to 2017-09-30
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.
We employed X-ray crystallography to build an atomic model of Shewanella denitrificans Kef C-terminal domain (SdKefQCTD) (PDB ID: 5NC8), which was used as model system to study Kef activation in various biophysical assays. Using this Kef model in conjunction with mutagenic studies, native mass spectrometry, high-performance liquid chromatography (HPLC), ligand-observed nuclear magnetic resonance (NMR) spectroscopy, molecular biology, different scanning fluorimetry, and molecular dynamics (MD) simulations, we elucidated the structural role of adenosine monophosphate (AMP) played in stabilizing the interfacial ligand-binding site of dimeric Kef protein. This work was recently published in Biochemistry 2017, 56 (32), pp 4219–4234.
Our previous work indicates that phenylalanine residue 441 (F441) in Escherichia coli KefC 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 KefC soluble C-terminal domain (EcKefCCTD) and biophysical studies conducted on SdKef soluble C-terminal domain (SdKefQCTD). 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. This fellowship project was set to investigate the dynamic mechanism of Kef activation using protein-observed 19F NMR (PrOF-NMR) spectroscopy. The F448 residue of SdKefQCTD was first site-specifically replaced by p-trifluoromethyl-L-phenylalanine (ptfmF) using amber stop codon suppression technology to produce a fluoro-Kef construct, SdKefQCTD(F448ptfmF). This enabled the dynamic movement of the 19F-labelled residue (F448ptfmF) to be monitored by PrOF-NMR upon binding to Kef ligands. We found that only Kef activators but not inhibitors can dynamically displace this gating residue. This confirmed that the large GSH adducts in Kef activator can clash with F448, evoking a significant conformational change in the protein, which ultimately leads to channel activation. These PrOF-NMR data proved for the first time that F448 is the gating residue and displacement of it leading to Kef activation. This PrOF-NMR method provides insights into both binding and function of Kef ligands, and therefore this technique should facilitate future discovery of potential antibiotics that target Kef. The results from this part of the project were already written in a manuscript draft, which will be submitted for peer-review in a scientific journal in the near future. The results of this part were also presented during a poster session in an academic conference.
In the last part of the project, E. coli kef knockout mutant expressing recombinant SdKef was employed as a bacterial model to test for its antibiotic susceptibility using Kirby-Bauer disk diffusion assay. We found that Kef activators can kill/inhibit the E. coli model. These data might pay a way to the future development of Kef-targeting antibacterials. The data of this part of the fellowship project will be published in a third paper in the future.
Our previous work indicates that phenylalanine residue 441 (F441) in Escherichia coli KefC 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 KefC soluble C-terminal domain (EcKefCCTD) and biophysical studies conducted on SdKef soluble C-terminal domain (SdKefQCTD). 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. This fellowship project was set to investigate the dynamic mechanism of Kef activation using protein-observed 19F NMR (PrOF-NMR) spectroscopy. The F448 residue of SdKefQCTD was first site-specifically replaced by p-trifluoromethyl-L-phenylalanine (ptfmF) using amber stop codon suppression technology to produce a fluoro-Kef construct, SdKefQCTD(F448ptfmF). This enabled the dynamic movement of the 19F-labelled residue (F448ptfmF) to be monitored by PrOF-NMR upon binding to Kef ligands. We found that only Kef activators but not inhibitors can dynamically displace this gating residue. This confirmed that the large GSH adducts in Kef activator can clash with F448, evoking a significant conformational change in the protein, which ultimately leads to channel activation. These PrOF-NMR data proved for the first time that F448 is the gating residue and displacement of it leading to Kef activation. This PrOF-NMR method provides insights into both binding and function of Kef ligands, and therefore this technique should facilitate future discovery of potential antibiotics that target Kef. The results from this part of the project were already written in a manuscript draft, which will be submitted for peer-review in a scientific journal in the near future. The results of this part were also presented during a poster session in an academic conference.
In the last part of the project, E. coli kef knockout mutant expressing recombinant SdKef was employed as a bacterial model to test for its antibiotic susceptibility using Kirby-Bauer disk diffusion assay. We found that Kef activators can kill/inhibit the E. coli model. These data might pay a way to the future development of Kef-targeting antibacterials. The data of this part of the fellowship project will be published in a third paper in the future.
This research project studied the bacterial potassium efflux system, Kef. Ultimately, this work might lead to the validation of Kef as a novel biological target for the development of a new antibiotic drug. The data generated from this work will be useful for other European researchers in the area of antibiotic resistance and antibiotic drug discovery. We will make the fluoro-Kef proteins, ligands, and 19F NMR techniques developed in this work available to the community to enable their studies. If we validate a novel antibiotic target and novel compounds that act as antibiotic drugs, our work will be of significant benefit to the European pharmaceutical industry, as it will underpin the discovery of novel antibiotic drugs. These drugs will contribute significantly to the European economy in terms of providing profit, employment, and investment of international pharmaceutical companies in Europe. In addition, as antibiotic resistance is a significant problem, drugs with novel modes of action will have a large impact on quality of public health.