The Role of divalent transition Metal ions in the binding of Fur dimer to DNA: Binding of Mn and Co to EC Fur dimer-DNA complex

Ferric uptake regulation protein is a repressor protein which binds an AT rich region of DNA (the iron box). Fur binds as a dimer in a helix turn helix mode and it is activated by iron(II) and other transition metal ions at elevated concentrations. Each transition metal ion induces certain conformational changes to aid the Fur binding, both the N-terminal and C-terminal domains take part in binding to DNA in addition to His 88 and His 86. The process is discussed in view of experimental reports. Fe(II), Mn(II) and Co(II) activate Fur to bind DNA but Zinc plays a structural role and does not activate Fur to bind DNA.


Introduction
Fur protein regulates the iron uptake in living cells by binding a 19-bp TATA called the iron or Fur box(1) (2). In previous work (3) (4) (5) (6), we have established that Fur dimer binds DNA in the presence of divalent metal ions as co-repressors (3), Fur protein employs the helix turn helix (HTH) mode in its DNA binding and using Fe 2+ as co-repressor in the biological systems to bind the DNA as a dimer (2). Experimentally, Fe 2+ , Co 2+ (7) and Mn 2+ acted as corepressors to activate Fur binding to the DNA(2) while Zn 2+ , Fe 3+ , Cd 2+ failed to activate Fur binding(2) (8). Zn 2+ ion plays a structural role (9)(10) (11), Fur dimer isolated from cells was reported to contain a structural zinc ion (12), recently the crystal structure of Mur tetramer with 8 zinc ions (2ions/Fur) (13). The crystal structure of a Fur dimer with 11Mn 2+ ions and 12 Zn 2+ ions was reported (14). indeed, we reported a first zinc ion site in which zinc ion is bound to C92, C95, H140 and Asp 137 (9,15) and this is recognized as the zinc site, there are no reported crystal structures with Fur bound to DNA. This work is aimed to give more insight on the crucial role of different transition metal ions (as co-repressors) and the ability of HTH in DNA binding proteins to accommodate more than one type of transition metal ion in their designed pockets (metal ion sites), at the same time, to make these ions capable of activating the repressor protein to bind specific DNA target.
Is there an effect for varying the divalent transition metal ion on the process of Fur binding to DNA and the role each metal ion plays in the conformational changes that take place in both Fur dimer and DNA in order to enhance the DNA binding process? An important aspect which will be discussed further in this study to help in understanding the vital role for metal ions in this process, does the metal ion play a structural role only, or does it have other functions to perform to make the specific binding successful.
Fur, being a global repressor activator protein capable of binding many genes. This gave the Fur repressor the flexibility to be activated by more than one transition metal ion in addition to its naturally occurring co- The study was extended to Mn (II), Co (II) binding in order to shed more light on this binding process and to obtain more information on the metal ion sensing process and the structural role metal ions play in the process as a whole.
Mn(II) is always used experimentally to replace the naturally occurring co-repressor ion Fe(II), both in vivo and in vitro due to its stability over Fe(II) and ease of handling because Fe(II) easily oxidizes, in addition to the role Mn(II) plays in biological processes which involves Fur binding to DNA.
The secrets of metal ion role in protein binding to DNA are revealed in this paper which will aid in better understanding of the role of metal ions in the DNA binding process (19), it becomes more established that metal ions play a structural role in addition to aiding the tuning mechanism of the protein dimer to a perfect, but not lasting fit on the DNA sequence. It is worth mentioning that none of the Fur crystal structures reported shows its DNA binding.

Computational Methods
All the molecular dynamics simulations(MD) were performed using AMBER molecular simulation package (20) . AMBER force field was used for molecular minimization and molecular dynamics. The analyses of MD trajectories were also preformed using AMBER. Pymol molecular viewer package was used for visualization (21).

Homology modeling of Fur protein
The known Fur sequence (from E. coli) was submitted to different modeler servers in order to predict the three-dimensional structure. SWISS MODEL

Results and Discussion
Proteolytic enzymes were used to detect metal-induced conformational changes in the ferric uptake regulation (      have crystal field stabilization energies which contributes to the binding and consequently to DNA binding of Fur upon conformational changes in the dimer. The ability of Fe 2+ , Co 2+ and Mn 2+ to bind the histidine nitrogen and aspartate oxygen plays a role in the co-repressor activity and to produce enough conformational change in the Fur dimer helices to shift the protein closer to DNA Fur box (Figure 1). Fe 2+ and Co 2+ ions proved to associate in larger quantities with Fur dimer up to 6:1(7), but maintaining the presence of the two major distorted sites(9)(3). The zinc ion proved to be a weak corepressor for Fur compared to the other ions , but its binding is enough for Arg112-Arg112   Table   1) The metal ligand distances agree well with our previous experimental work