| Iron is an essential element in microbial metabolism,playing a crucial role as a cofactor in various life processes.Despite its abundance in nature,microorganisms can only utilize a soluble Fe(Ⅱ)content of 10-18 M due to the extremely low solubility of Fe(Ⅲ)in oxygen-rich and water-rich environments.Consequently,iron competition becomes a vital aspect of microbial habitat adaptation.Although iron is necessary for microbial survival,excessive Fe(Ⅱ)can induce cellular damage through oxidative stress.Hence,microorganisms must tightly regulate intracellular iron concentration to maintain iron homeostasis.E.coli has developed an efficient and complex mechanism for iron homeostasis regulation,with the ferric uptake regulator protein(Fur)playing a crucial role.Fur is a key player in iron homeostasis,binding to the promoter regions of iron metabolism genes and controlling their transcription upon sensing iron.Since its discovery in E.coli in 1981,over 16,000 homologous proteins have been reported.The ability of Fur to bind its target DNA relies on the protein’s conformation,which,in turn,is influenced by the type,quantity,and location of metal ions involved in binding.Currently,there are significant controversies surrounding the conformation and ion binding of Fur in E.coli.In this study,we have conducted a comprehensive analysis of the full-length structure of Fur in E.coli,providing mechanistic insights and a structural foundation for investigating iron-induced Fur activation and DNA recognition.The main objectives of this thesis are as follows:(1)Determination of the full-length structure of E.coli Fur at a resolution of 2.57 (?).The classical ion regulation model of Fur was combined with a novel regulatory model involving YdiV-Fur-SlyD.Fur expression was achieved by knocking out slyD,followed by dialysis and incubation with metal ions.Through crystal screening and optimization,crystallographic data at 2.57 A resolution was obtained.The structure revealed the binding of three metal ions to each monomer:ion binding site 1(H133,E81,H88,H90),site 2(H87,D89,E108,H125),and site 3(C93,C96,C138,H145).(2)Each monomer of Fur combines two irons and one zincProtein variants with mutations in different metal ion binding sites were obtained through amino acid site-directed mutagenesis.The binding of iron ions at site 1 and site 2,as well as the binding of a zinc ion at site 3,was determined using ICP-MS.(3)The effect of ion binding on Fur’s DNA binding function was verified in vitroITC experiments demonstrated weakened ion binding ability of Fur protein variants with mutations in ion binding sites.EMSA and FP experiments were performed to evaluate the binding capacity of the mutated Fur proteins to Fur box DNA.Compared to wild-type Fur,mutations in the iron ion binding sites(site 1 and 2)significantly reduced the protein’s DNA binding ability,while mutations in the zinc ion binding site(site 3)did not exhibit a noticeable effect on DNA binding.(4)The disturbance caused by mutations in Fur ion binding sites on the activation of iron metabolism genes during iron deprivation was validated in vivo.Site-directed mutations were introduced in the genome of E.coli wild-type strain MG1655,targeting the three metal ion binding sites.ICP-MS analysis confirmed a decrease in total cellular iron content.qRT-PCR analysis revealed time-dependent expression of Fur-regulated genes in response to iron deficiency,and the mutations in ion binding sites perturbed the expression of iron uptake genes regulated by Fur to varying degrees.Notably,the mutation in the zinc ion binding site resulted in almost no response to iron-deficiency signals in iron uptake genes.In conclusion,this study focused on the structure and function of the iron regulatory protein Fur in E.coli.The full-length crystal structure of E.coli Fur was determined,elucidating its ion binding characteristics and actions.Additionally,the temporal regulation of iron metabolism genes by Fur in E.coli was explored.This research provides an important contribution to the understanding of microbial iron metabolism. |
