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.
