Conserved cysteine residues are necessary for nickel-induced allosteric regulation of the metalloregulatory protein YqjI (NfeR) in E. coli

• Published in: Journal of inorganic biochemistry Vol. 184; pp. 123 - 133
• Main Authors: Blahut, Matthew, Dzul, Stephen, Wang, Suning, Kandegedara, Ashoka, Grossoehme, Nicholas E., Stemmler, Timothy, Outten, F. Wayne
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.2018
• Subjects: EXAFS; Metal homeostasis; Nickel; Vicinal disulfide; YqjH; YqjI; Vicinal disulfide; Nickel; YqjH; Metal homeostasis; YqjI; EXAFS
• ISSN: 0162-0134; 1873-3344
• DOI: 10.1016/j.jinorgbio.2018.04.016

Transition metal homeostasis is necessary to sustain life. First row transition metals act as cofactors within the cell, performing vital functions ranging from DNA repair to respiration. However, intracellular metal concentrations exceeding physiological requirements may be toxic. In E. coli, the YqjH flavoprotein is thought to play a role in iron homeostasis. YqjH is transcriptionally regulated by the ferric uptake regulator and a newly discovered regulator encoded by yqjI. The apo-form of YqjI is a transcriptional repressor of both the yqjH and yqjI genes. YqjI repressor function is disrupted upon binding of nickel. The YqjI N-terminus is homologous to nickel-binding proteins, implicating this region as a nickel-binding domain. Based on function, yqjI and yqjH should be renamed Ni-responsive Fe-uptake regulator (nfeR) and Ni-responsive Fe-uptake flavoprotein (nfeF), respectively. X-ray Absorption Spectroscopy was employed to characterize the nickel binding site(s) within YqjI. Putative nickel binding ligands were targeted by site-directed mutagenesis and resulting variants were analyzed in vivo for repressor function. Isothermal titration calorimetry and competitive binding assays were used to further quantify nickel interactions with wild-type YqjI and its mutant derivatives. Results indicate plasticity in the nickel binding domain of YqjI. Residues C42 and C43 were found to be required for in vivo response of YqjI to nickel stress, though these residues are not required for in vitro nickel binding. We propose that YqjI may contain a vicinal disulfide bond between C42 and C43 that is important for nickel-responsive allosteric interactions between YqjI domains. The unstructured N-terminus of the metalloregulatory protein YqjI is rich in metal-binding residues but specifically requires two conserved cysteine residues to coordinate nickel-binding with negative allosteric regulation of the DNA-binding domain, thereby inhibiting the YqjI transcriptional repressor function. Based on its function, yqjI should be renamed Ni-responsive Fe-uptake regulator, nfeR. [Display omitted] •Native metalloregulatory protein YqjI binds two nickel ions with low nanomolar affinity.•Based on its function, yqjI should be renamed Ni-responsive Fe-uptake regulator, nfeR.•Cys42 and 43 are required for nickel-dependent control of YqjI.•Cys42 and Cys43 are not obligate ligands for nickel binding to YqjI.•Cys42 and Cys43 may be in a vicinal disulfide important for YqjI regulation.