Quantitative analysis of the binding between the master bacterial iron regulator and a target DNA sequence

Abstract

Iron is an essential micronutrient that is utilized by bacteria for numerous cellular and metabolic processes. The main way bacteria use to regulate iron homeostasis is via transcription regulation mediated by the ferric uptake regulator (Fur). In the presence of excess iron, the Fur protein acts as a transcriptional repressor of genes necessary for iron regulation. When the Fur protein binds to Fe2+, the Fur‐Fe2+ complex will bind to a target DNA sequence named the Fur box, which is located within the promoter of Fur‐regulated genes. However, when the iron level is limited, Fur dissociates from the Fur box and allows gene transcription. The Fur system, which is highly conserved throughout the different genera of bacteria, is absent in eukaryotes. Therefore, this unique feature of the Fur system combined with the essential nature of metal homeostasis in bacteria makes Fur a promising target for antimicrobials.Previous lab results identified a set of highly conserved residues in the Klebsiella sp. Fur protein, and site‐directed mutagenesis was carried out to obtain point mutants. β‐galactosidase assays were conducted to determine which mutants were affected in the in vivo and in vitro interaction between the Fur protein and a target DNA sequence (Fur box). Three mutants, H88A, H90A, and C96S, were found to exhibit a significantly higher level of LacZ activity, indicating that these are critical residues that affect the binding affinity to the DNA.In this work, the three mutant Fur proteins were overexpressed in E. coli BL21 cells and purified via affinity chromatography. A GST‐Fur protein was first purified using a GSH‐Sepharose resin, followed by cleavage of the GST tag and an additional purification step on a Heparin‐Sepharose resin. SDS‐PAGE analysis was used to identify fractions containing the purified recombinant proteins. In order to further examine the binding between Fur and the target DNA sequence, our lab applied BLItz biolayer interferometry. BLItz system is a direct binding assay that takes place on a biosensor probe. The biosensor surface only detects the molecules that bind directly to the probe, allowing us to determine the binding specificity and the dissociation constant (Kd). We obtained a commercially available streptavidin biosensor, which has a strong affinity for biotin‐labeled DNA (biotin‐labeled Fur box sequence). This allowed us to measure the binding constant between the Fur box and Fur protein by exposing the biotin‐labeled Fur box‐bound biosensor to the Fur protein.Among the wild‐type (WT), H88A, H90A, and C96S Fur proteins, the WT Fur protein showed significantly stronger binding affinity with the DNA compared to that of other three point‐mutant Fur proteins. These quantitative measures demonstrate that H88, H90, and C96 are indeed important residues that play major role in the binding affinity between the Fur protein and DNA binding.Support or Funding InformationThis project was supported by Midwestern University intramural funds. Samuel Cheong was a Kenneth A. Suarez summer research fellow at Midwestern University.